Fission Yeast as a Platform for Antibacterial Drug Screens Targeting Bacterial Cytoskeleton Proteins.

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB have emerged as important targets for novel antimicrobial discovery. Several assays have been developed to identify compounds targeting nucleotide binding and polymerization of these cytoskeletal proteins, primarily focused on FtsZ. Moreover, many of the assays are either laborious or cost-intensive, and ascertaining whether these proteins are the cellular target of the drug often requires multiple methods. Finally, the toxicity of the drugs to eukaryotic cells also poses a problem. Here, we describe a single-step cell-based assay to discover novel molecules targeting bacterial cytoskeleton and minimize hits that might be potentially toxic to eukaryotic cells. Fission yeast is amenable to high-throughput screens based on microscopy, and a visual screen can easily identify any molecule that alters the polymerization of FtsZ or MreB. Our assay utilizes the standard 96-well plate and relies on the ability of the bacterial cytoskeletal proteins to polymerize in a eukaryotic cell such as the fission yeast. While the protocols described here are for fission yeast and utilize FtsZ from Staphylococcus aureus and MreB from Escherichia coli, they are easily adaptable to other bacterial cytoskeletal proteins that readily assemble into polymers in any eukaryotic expression hosts. The method described here should help facilitate further discovery of novel antimicrobials targeting bacterial cytoskeletal proteins.

Chromodomain mutation in S. pombe Kat5/Mst1 affects centromere dynamics and DNA repair.

KAT5 (S. pombe Mst1, human TIP60) is a MYST family histone acetyltransferase conserved from yeast to humans that is involved in multiple cellular activities. This family is characterized in part by containing a chromodomain, a motif associated with binding methylated histones. We show that a chromodomain mutation in the S. pombe Kat5, mst1-W66R, has defects in pericentromere silencing. mst1-W66R is sensitive to camptothecin (CPT) but only at an increased temperature of 36°C, although it is proficient for growth at this temperature. We also describe a de-silencing effect at the pericentromere by CPT that is independent of RNAi and methylation machinery. We also show that mst1-W66R disrupts recruitment of proteins to repair foci in response to camptothecin-induced DNA damage. Our data suggest a function of Mst1 chromodomain in centromere heterochromatin formation and a separate role in genome-wide damage repair in CPT.

Low-Molecular Weight Compounds that Extend the Chronological Lifespan of Yeasts, Saccharomyces cerevisiae, and Schizosaccharomyces pombe.

Yeast is an excellent model organism for research for regulating aging and lifespan, and the studies have made many contributions to date, including identifying various factors and signaling pathways related to aging and lifespan. More than 20 years have passed since molecular biological perspectives are adopted in this research field, and intracellular factors and signal pathways that control aging and lifespan have evolutionarily conserved from yeast to mammals. Furthermore, these findings have been applied to control the aging and lifespan of various model organisms by adjustment of the nutritional environment, genetic manipulation, and drug treatment using low-molecular weight compounds. Among these, drug treatment is easier than the other methods, and research into drugs that regulate aging and lifespan is consequently expected to become more active. Chronological lifespan, a definition of yeast lifespan, refers to the survival period of a cell population under nondividing conditions. Herein, low-molecular weight compounds are summarized that extend the chronological lifespan of Saccharomyces cerevisiae and Schizosaccharomyces pombe, along with their intracellular functions. The low-molecular weight compounds are also discussed that extend the lifespan of other model organisms. Compounds that have so far only been studied in yeast may soon extend lifespan in other organisms.

Ellagic Acid Combined with Tacrolimus Showed Synergistic Cell Growth Inhibition in Fission Yeast.

Calcineurin (CN) is a conserved Ca2+-calmodulin activated protein phosphatase, which plays important roles in immune regulation, cardiac hypertrophy, and apoptosis in humans. In pathogenic fungi, CN is essential for stress survival, sexual development, and virulence. The immunosuppressant tacrolimus (FK506) is a specific inhibitor of CN in humans and fungi including nonpathogenic fission yeast. Although calcineurin inhibition by FK506 or CN deletion in fission yeast does not induce growth defects, treatment with some anti-fungal drugs such as micafungin and valproic acid, induced synthetic lethality with calcineurin inhibition. Here, we searched for the compounds that induce synthetic growth defects with CN inhibition in fission yeast. We found that ellagic acid (EA) preferentially induced growth inhibition in CN deletion cells. Consistently, co-treatment with EA and FK506 induced severe growth inhibition in the wild-type cells, whereas neither of the single treatment with each compound did so. Moreover, deletion of the calcineurin-regulated transcription factor Prz1 also induced a marked EA sensitivity. Intriguingly, EA also enhanced the growth inhibitory effect of other anti-fungal drugs, including micafungin and miconazole. Thus, our data suggesting the synergistic growth inhibitory effect of the calcineurin inhibitor FK506 and EA may be useful to understand the mechanism to overcome the antifungal resistance.

Cytokinin Inhibits Fungal Development and Virulence by Targeting the Cytoskeleton and Cellular Trafficking.

Cytokinin (CK) is an important plant developmental regulator, having activities in many aspects of plant life and response to the environment. CKs are involved in diverse processes in the plant, including stem cell maintenance, vascular differentiation, growth and branching of roots and shoots, leaf senescence, nutrient balance, and stress tolerance. In some cases, phytopathogens secrete CKs. It has been suggested that to achieve pathogenesis in the host, CK-secreting biotrophs manipulate CK signaling to regulate the host cell cycle and nutrient allocation. CK is known to induce host plant resistance to several classes of phytopathogens from a few works, with induced host immunity via salicylic acid signaling suggested to be the prevalent mechanism for this host resistance. Here, we show that CK directly inhibits the growth, development, and virulence of fungal phytopathogens. Focusing on Botrytis cinerea (Bc), we demonstrate that various aspects of fungal development can be reversibly inhibited by CK. We also found that CK affects both budding and fission yeast in a similar manner. Investigating the mechanism by which CK influences fungal development, we conducted RNA next-generation sequencing (RNA-NGS) on mock- and CK-treated B. cinerea samples, finding that CK alters the cell cycle, cytoskeleton, and endocytosis. Cell biology experiments demonstrated that CK affects cytoskeleton components and cellular trafficking in Bc, lowering endocytic rates and endomembrane compartment sizes, likely leading to reduced growth rates and arrested developmental programs. Mutant analyses in yeast confirmed that the endocytic pathway is altered by CK. Our work uncovers a remarkably conserved role for a plant growth hormone in fungal biology, suggesting that pathogen-host interactions resulted in fascinating molecular adaptations on fundamental processes in eukaryotic biology. IMPORTANCE Cytokinins (CKs), important plant growth/developmental hormones, have previously been associated with host disease resistance. Here, we demonstrate that CK directly inhibits the growth, development, and virulence of B. cinerea (Bc) and many additional phytopathogenic fungi. Molecular and cellular analyses revealed that CK is not toxic to Bc, but rather, Bc likely recognizes CK and responds to it, resulting in cell cycle and individual cell growth retardation, via downregulation of cytoskeletal components and endocytic trafficking. Mutant analyses in yeast confirmed that the endocytic pathway is a CK target. Our work demonstrates a conserved role for CK in yeast and fungal biology, suggesting that pathogen-host interactions may cause molecular adaptations in fundamental processes in eukaryotic biology.

A rapidly reversible mutation generates subclonal genetic diversity and unstable drug resistance.

Most genetic changes have negligible reversion rates. As most mutations that confer resistance to an adverse condition (e.g., drug treatment) also confer a growth defect in its absence, it is challenging for cells to genetically adapt to transient environmental changes. Here, we identify a set of rapidly reversible drug-resistance mutations in Schizosaccharomyces pombe that are caused by microhomology-mediated tandem duplication (MTD) and reversion back to the wild-type sequence. Using 10,000× coverage whole-genome sequencing, we identify nearly 6,000 subclonal MTDs in a single clonal population and determine, using machine learning, how MTD frequency is encoded in the genome. We find that sequences with the highest-predicted MTD rates tend to generate insertions that maintain the correct reading frame, suggesting that MTD formation has shaped the evolution of coding sequences. Our study reveals a common mechanism of reversible genetic variation that is beneficial for adaptation to environmental fluctuations and facilitates evolutionary divergence.

Identification of novel microtubule inhibitors effective in fission yeast and human cells and their effects on breast cancer cell lines.

Microtubules are critical for a variety of cellular processes such as chromosome segregation, intracellular transport and cell shape. Drugs against microtubules have been widely used in cancer chemotherapies, though the acquisition of drug resistance has been a significant issue for their use. To identify novel small molecules that inhibit microtubule organization, we conducted sequential phenotypic screening of fission yeast and human cells. From a library of diverse 10 371 chemicals, we identified 11 compounds that inhibit proper mitotic progression both in fission yeast and in HeLa cells. An in vitro assay revealed that five of these compounds are strong inhibitors of tubulin polymerization. These compounds directly bind tubulin and destabilize the structures of tubulin dimers. We showed that one of the compounds, L1, binds to the colchicine-binding site of microtubules and exhibits a preferential potency against a panel of human breast cancer cell lines compared with a control non-cancer cell line. In addition, L1 overcomes cellular drug resistance mediated by ßIII tubulin overexpression and has a strong synergistic effect when combined with the Plk1 inhibitor BI2536. Thus, we have established an economically effective drug screening strategy to target mitosis and microtubules, and have identified a candidate compound for cancer chemotherapy.

A squalene-hopene cyclase in Schizosaccharomyces japonicus represents a eukaryotic adaptation to sterol-limited anaerobic environments.

Biosynthesis of sterols, which are key constituents of canonical eukaryotic membranes, requires molecular oxygen. Anaerobic protists and deep-branching anaerobic fungi are the only eukaryotes in which a mechanism for sterol-independent growth has been elucidated. In these organisms, tetrahymanol, formed through oxygen-independent cyclization of squalene by a squalene-tetrahymanol cyclase, acts as a sterol surrogate. This study confirms an early report [C. J. E. A. Bulder, Antonie Van Leeuwenhoek, 37, 353-358 (1971)] that Schizosaccharomyces japonicus is exceptional among yeasts in growing anaerobically on synthetic media lacking sterols and unsaturated fatty acids. Mass spectrometry of lipid fractions of anaerobically grown Sch. japonicus showed the presence of hopanoids, a class of cyclic triterpenoids not previously detected in yeasts, including hop-22(29)-ene, hop-17(21)-ene, hop-21(22)-ene, and hopan-22-ol. A putative gene in Sch. japonicus showed high similarity to bacterial squalene-hopene cyclase (SHC) genes and in particular to those of Acetobacter species. No orthologs of the putative Sch. japonicus SHC were found in other yeast species. Expression of the Sch. japonicus SHC gene (Sjshc1) in Saccharomyces cerevisiae enabled hopanoid synthesis and stimulated anaerobic growth in sterol-free media, thus indicating that one or more of the hopanoids produced by SjShc1 could at least partially replace sterols. Use of hopanoids as sterol surrogates represents a previously unknown adaptation of eukaryotic cells to anaerobic growth. The fast anaerobic growth of Sch. japonicus in sterol-free media is an interesting trait for developing robust fungal cell factories for application in anaerobic industrial processes.

The intra-S phase checkpoint directly regulates replication elongation to preserve the integrity of stalled replisomes.

DNA replication is dramatically slowed down under replication stress. The regulation of replication speed is a conserved response in eukaryotes and, in fission yeast, requires the checkpoint kinases Rad3ATR and Cds1Chk2 However, the underlying mechanism of this checkpoint regulation remains unresolved. Here, we report that the Rad3ATR-Cds1Chk2 checkpoint directly targets the Cdc45-MCM-GINS (CMG) replicative helicase under replication stress. When replication forks stall, the Cds1Chk2 kinase directly phosphorylates Cdc45 on the S275, S322, and S397 residues, which significantly reduces CMG helicase activity. Furthermore, in cds1Chk2 -mutated cells, the CMG helicase and DNA polymerases are physically separated, potentially disrupting replisomes and collapsing replication forks. This study demonstrates that the intra-S phase checkpoint directly regulates replication elongation, reduces CMG helicase processivity, prevents CMG helicase delinking from DNA polymerases, and therefore helps preserve the integrity of stalled replisomes and replication forks.

Genome-wide screening of genes associated with momilactone B sensitivity in the fission yeast Schizosaccharomyces pombe.

Momilactone B is a natural product with dual biological activities, including antimicrobial and allelopathic properties, and plays a major role in plant chemical defense against competitive plants and pathogens. The pharmacological effects of momilactone B on mammalian cells have also been reported. However, little is known about the molecular and cellular mechanisms underlying its broad bioactivity. In this study, the genetic determinants of momilactone B sensitivity in yeast were explored to gain insight into its mode of action. We screened fission yeast mutants resistant to momilactone B from a pooled culture containing genome-wide gene-overexpressing strains in a drug-hypersensitive genetic background. Overexpression of pmd1, bfr1, pap1, arp9, or SPAC9E9.06c conferred resistance to momilactone B. In addition, a drug-hypersensitive, barcoded deletion library was newly constructed and the genes that imparted altered sensitivity to momilactone B upon deletion were identified. Gene Ontology and fission yeast phenotype ontology enrichment analyses predicted the biological pathways related to the mode of action of momilactone B. The validation of predictions revealed that momilactone B induced abnormal phenotypes such as multiseptated cells and disrupted organization of the microtubule structure. This is the first investigation of the mechanism underlying the antifungal activity of momilactone B against yeast. The results and datasets obtained in this study narrow the possible targets of momilactone B and facilitate further studies regarding its mode of action.

Dual Impact of a Benzimidazole Resistant ß-Tubulin on Microtubule Behavior in Fission Yeast.

The cytoskeleton microtubule consists of polymerized αß-tubulin dimers and plays essential roles in many cellular events. Reagents that inhibit microtubule behaviors have been developed as antifungal, antiparasitic, and anticancer drugs. Benzimidazole compounds, including thiabendazole (TBZ), carbendazim (MBC), and nocodazole, are prevailing microtubule poisons that target ß-tubulin and inhibit microtubule polymerization. The molecular basis, however, as to how the drug acts on ß-tubulin remains controversial. Here, we characterize the S. pombe ß-tubulin mutant nda3-TB101, which was previously isolated as a mutant resistance to benzimidazole. The mutation site tyrosine at position 50 is located in the interface of two lateral ß-tubulin proteins and at the gate of a putative binging pocket for benzimidazole. Our observation revealed two properties of the mutant tubulin. First, the dynamics of cellular microtubules comprising the mutant ß-tubulin were stabilized in the absence of benzimidazole. Second, the mutant protein reduced the affinity to benzimidazole in vitro. We therefore conclude that the mutant ß-tubulin Nda3-TB101 exerts a dual effect on microtubule behaviors: the mutant ß-tubulin stabilizes microtubules and is insensitive to benzimidazole drugs. This notion fine-tunes the current elusive molecular model regarding binding of benzimidazole to ß-tubulin.

Construction and characterization of a zinc-inducible gene expression vector in fission yeast.

Gene expression vectors are useful and important tools that are commonly used in a variety of experiments, including expression of foreign genes, functional analysis of genes of interest and complementation experiments. In this study, a hybrid promoter, combining the adh1+ upstream activating sequence (UAS) of fission yeast and the GAL10 core promoter of budding yeast, was constructed to enable high level expression depending on the presence of zinc in culture medium for fission yeast. When the hybrid promoter was cloned on the multicopy plasmid, it was fully induced and repressed within 10 h in the presence and absence of zinc, respectively. The kinetics of induction and reduction were similar to those of the endogenous adh1+ mRNA. In contrast, native adh1+ promoter lost its tight repression in zinc-depleted condition when it was cloned on the plasmid. Because adh1+ UAS-specific transcription factors have not yet been identified, we identified UAS elements involved in zinc sensing by characterizing this hybrid promoter. We also found that the expression level increased by the TATA box mutation, GATAA, in the presence of zinc.

Ribosome profiling reveals ribosome stalling on tryptophan codons and ribosome queuing upon oxidative stress in fission yeast.

Translational control is essential in response to stress. We investigated the translational programmes launched by the fission yeast Schizosaccharomyces pombe upon five environmental stresses. We also explored the contribution of defence pathways to these programmes: The Integrated Stress Response (ISR), which regulates translation initiation, and the stress-response MAPK pathway. We performed ribosome profiling of cells subjected to each stress, in wild type cells and in cells with the defence pathways inactivated. The transcription factor Fil1, a functional homologue of the yeast Gcn4 and the mammalian Atf4 proteins, was translationally upregulated and required for the response to most stresses. Moreover, many mRNAs encoding proteins required for ribosome biogenesis were translationally downregulated. Thus, several stresses trigger a universal translational response, including reduced ribosome production and a Fil1-mediated transcriptional programme. Surprisingly, ribosomes stalled on tryptophan codons upon oxidative stress, likely due to a decrease in charged tRNA-Tryptophan. Stalling caused ribosome accumulation upstream of tryptophan codons (ribosome queuing/collisions), demonstrating that stalled ribosomes affect translation elongation by other ribosomes. Consistently, tryptophan codon stalling led to reduced translation elongation and contributed to the ISR-mediated inhibition of initiation. We show that different stresses elicit common and specific translational responses, revealing a novel role in Tryptophan-tRNA availability.

Hypoosmolality impedes cytoophidium integrity during nitrogen starvation.

CTP synthase (CTPS) cytoophidia have been found in many species over domains of life in the past 10 years, implying the evolutionary conservation of these structures. However, there are differences in cytoophidia between species. The difference in CTPS cytoophidium properties between budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) inspires this research. We study the effects of culture environment on cytoophidia in S. cerevisiae by switching to the optimal medium for S. pombe. S. cerevisiae CTPS cytoophidium fragmentation and pseudohyphae formation are observed after treatment with S. pombe medium YES instead of S. cerevisiae medium YPD. By modifying the level of each ingredient of the media, we find that hypoosmolality impedes cytoophidium integrity during nitrogen starvation. Our study demonstrates the relationship between cytoophidium integrity and environmental stress, supporting the role of cytoophidia in stress resistance.

Molecular Diversity via Tetrasubstituted Alkenes Containing a Barbiturate Motif: Synthesis and Biological Activity.

The synthesis of a molecularly diverse library of tetrasubstituted alkenes containing a barbiturate motif is described. Base-induced condensation of N1-substituted pyrimidine-2,4,6(1H,3H,5H)-triones with 5-(bis(methylthio)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione gave 3-substituted 5-(methylthio)-2H-pyrano[2,3-d]pyrimidine-2,4,7(1H,3H)-triones ('pyranopyrimidinones'), regioselectively. A sequence of reactions involving ring-opening of the pyran moiety, displacement of the methylthio group with an amine, re-formation of the pyran ring, and after its final cleavage with an amine, gave tetrasubstituted alkenes (3-amino-3-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)propanamides) with a diversity of substituents. Cleavage of the pyranopyrimidinones with an aniline was facilitated in 2,2,2-trifluoroethanol under microwave irradiation. Compounds were tested against Escherichia coli, Staphylococcus aureus, the yeast Schizosaccharomyces pombe, and the pathogenic fungus Candida albicans. No compounds exhibited activity against E. coli, whilst one compound was weakly active against S. aureus. Three compounds were strongly active against S. pombe, but none was active against C. albicans.

Cadmium-Induced Cell Homeostasis Impairment is Suppressed by the Tor1 Deficiency in Fission Yeast.

Cadmium has no known physiological function in the body; however, its adverse effects are associated with cancer and many types of organ system damage. Although much has been shown about Cd toxicity, the underlying mechanisms of its responses to the organism remain unclear. In this study, the role of Tor1, a catalytic subunit of the target of rapamycin complex 2 (TORC2), in Cd-mediated effects on cell proliferation, the antioxidant system, morphology, and ionome balance was investigated in the eukaryotic model organism Schizosaccharomyces pombe. Surprisingly, spectrophotometric and biochemical analyses revealed that the growth rate conditions and antioxidant defense mechanisms are considerably better in cells lacking the Tor1 signaling. The malondialdehyde (MDA) content of Tor1-deficient cells upon Cd treatment represents approximately half of the wild-type content. The microscopic determination of the cell morphological parameters indicates the role for Tor1 in cell shape maintenance. The ion content, determined by inductively coupled plasma optical emission spectroscopy (ICP-OES), showed that the Cd uptake potency was markedly lower in Tor1-depleted compared to wild-type cells. Conclusively, we show that the cadmium-mediated cell impairments in the fission yeast significantly depend on the Tor1 signaling. Additionally, the data presented here suggest the yet-undefined role of Tor1 in the transport of ions.

Epigenetic gene silencing by heterochromatin primes fungal resistance.

Heterochromatin that depends on histone H3 lysine 9 methylation (H3K9me) renders embedded genes transcriptionally silent1-3. In the fission yeast Schizosaccharomyces pombe, H3K9me heterochromatin can be transmitted through cell division provided the counteracting demethylase Epe1 is absent4,5. Heterochromatin heritability might allow wild-type cells under certain conditions to acquire epimutations, which could influence phenotype through unstable gene silencing rather than DNA change6,7. Here we show that heterochromatin-dependent epimutants resistant to caffeine arise in fission yeast grown with threshold levels of caffeine. Isolates with unstable resistance have distinct heterochromatin islands with reduced expression of embedded genes, including some whose mutation confers caffeine resistance. Forced heterochromatin formation at implicated loci confirms that resistance results from heterochromatin-mediated silencing. Our analyses reveal that epigenetic processes promote phenotypic plasticity, letting wild-type cells adapt to unfavourable environments without genetic alteration. In some isolates, subsequent or coincident gene-amplification events augment resistance. Caffeine affects two anti-silencing factors: Epe1 is downregulated, reducing its chromatin association, and a shortened isoform of Mst2 histone acetyltransferase is expressed. Thus, heterochromatin-dependent epimutation provides a bet-hedging strategy allowing cells to adapt transiently to insults while remaining genetically wild type. Isolates with unstable caffeine resistance show cross-resistance to antifungal agents, suggesting that related heterochromatin-dependent processes may contribute to resistance of plant and human fungal pathogens to such agents.

Discovery of a Cryptic Depsipeptide from Streptomyces ghanaensis via MALDI-MS-Guided High-Throughput Elicitor Screening.

Microbial genomes harbor an abundance of biosynthetic gene clusters, but most are expressed at low levels and need to be activated for characterization of their cognate natural products. In this work, we report the combination of high-throughput elicitor screening (HiTES) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the rapid identification of cryptic peptide natural products. Application to Streptomyces ghanaensis identified amygdalin as an elicitor of a novel non-ribosomal peptide, which we term cinnapeptin. Complete structural elucidation revealed cinnapeptin as a cyclic depsipeptide with an unusual 2-methyl-cinnamoyl group. Insights into its biosynthesis were provided by whole genome sequencing and gene deletion studies, while bioactivity assays showed antimicrobial activity against Gram-positive bacteria and fission yeast. MALDI-HiTES is a broadly applicable tool for the discovery of cryptic peptides encoded in microbial genomes.

Chemical genetic analysis of FTY720- and Ca2+ -sensitive mutants reveals a functional connection between FTY720 and membrane trafficking.

FTY720, a sphingosine-1-phosphate (S1P) analog, is used as an immune modulator to treat multiple sclerosis. Accumulating evidence has suggested the mode of action of FTY720 independent of an S1P modulator. In fission yeast, FTY720 induces an increase in intracellular Ca2+ and ROS levels. We have previously identified 49 genes of which deletion causes FTY720 sensitivity. Here, we characterized the FTY720-sensitive mutants in terms of their relevance to the Ca2+ homeostasis and identified the 16 FTY720- and Ca2+ -sensitive mutants (fcs mutants). Most of the FTY720-sensitive mutants showed elevated Ca2+ levels and exhibited Ca2+ dysregulation by FTY720 treatment. One of the functional categories among the genes whose deletion renders cells susceptible to FTY720 and Ca2+ include the Golgi/endosomal membrane trafficking. Notably, FTY720, but not phosphorylated FTY720 incapable of inducing Ca2+ increase, inhibited the secretion of acid phosphatase in the wild-type cells. Importantly, secretory defects of the Golgi/endosomal trafficking mutants, Vps45, or Ryh1 deletion, were further exacerbated by FTY720. Our fcs mutant screen also identified the adenylyl cyclase-associated protein Cap1 and a Rictor homolog Ste20, whose deletion markedly exacerbated FTY720-sensitive secretory impairment. Collectively, our data may suggest a synergistic impact of FTY720 combined with secretion perturbation on proliferation and Ca2+ homeostasis.

Glucose limitation and pka1 deletion rescue aberrant mitotic spindle formation induced by Mal3 overexpression in Schizosaccharomyces pombe.

The cAMP-dependent protein kinase Pka1 is known as a regulator of glycogenesis, transition into meiosis, proper chromosome segregation, and stress responses in Schizosaccharomyces pombe. We demonstrated that both the cAMP/PKA pathway and glucose limitation play roles in appropriate spindle formation. Overexpression of Mal3 (1-308), an EB1 family protein, caused growth defects, increased 4C DNA content, and induced monopolar spindle formation. Overproduction of a high-affinity microtubule binding mutant (Q89R) and a recombinant protein possessing the CH and EB1 domains (1-241) both resulted in more severe phenotypes than Mal3 (1-308). Loss of functional Pka1 and glucose limitation rescued the phenotypes of Mal3-overexpressing cells, whereas deletion of Tor1 or Ssp2 did not. Growth defects and monopolar spindle formation in a kinesin-5 mutant, cut7-446, was partially rescued by pka1 deletion or glucose limitation. These findings suggest that Pka1 and glucose limitation regulate proper spindle formation in Mal3-overexpressing cells and the cut7-446 mutant.