Massive expression of cysteine-containing proteins causes abnormal elongation of yeast cells by perturbing the proteasome.

The enhanced green fluorescent protein (EGFP) is considered to be a harmless protein because the critical expression level that causes growth defects is higher than that of other proteins. Here, we found that overexpression of EGFP, but not a glycolytic protein Gpm1, triggered the cell elongation phenotype in the budding yeast Saccharomyces cerevisiae. By the morphological analysis of the cell overexpressing fluorescent protein and glycolytic enzyme variants, we revealed that cysteine content was associated with the cell elongation phenotype. The abnormal cell morphology triggered by overexpression of EGFP was also observed in the fission yeast Schizosaccharomyces pombe. Overexpression of cysteine-containing protein was toxic, especially at high-temperature, while the toxicity could be modulated by additional protein characteristics. Investigation of protein aggregate formation, morphological abnormalities in mutants, and transcriptomic changes that occur upon overexpression of EGFP variants suggested that perturbation of the proteasome by the exposed cysteine of the overexpressed protein causes cell elongation. Overexpression of proteins with relatively low folding properties, such as EGFP, was also found to promote the formation of SHOTA (Seventy kDa Heat shock protein-containing, Overexpression-Triggered Aggregates), an intracellular aggregate that incorporates Hsp70/Ssa1, which induces a heat shock response, while it was unrelated to cell elongation. Evolutionary analysis of duplicated genes showed that cysteine toxicity may be an evolutionary bias to exclude cysteine from highly expressed proteins. The overexpression of cysteine-less moxGFP, the least toxic protein revealed in this study, would be a good model system to understand the physiological state of protein burden triggered by ultimate overexpression of harmless proteins.

Pcp1/pericentrin controls the SPB number in fission yeast meiosis and ploidy homeostasis.

During sexual reproduction, the zygote must inherit exactly one centrosome (spindle pole body [SPB] in yeasts) from the gametes, which then duplicates and assembles a bipolar spindle that supports the subsequent cell division. Here, we show that in the fission yeast Schizosaccharomyces pombe, the fusion of SPBs from the gametes is blocked in polyploid zygotes. As a result, the polyploid zygotes cannot proliferate mitotically and frequently form supernumerary SPBs during subsequent meiosis, which leads to multipolar nuclear divisions and the generation of extra spores. The blockage of SPB fusion is caused by persistent SPB localization of Pcp1, which, in normal diploid zygotic meiosis, exhibits a dynamic association with the SPB. Artificially induced constitutive localization of Pcp1 on the SPB is sufficient to cause blockage of SPB fusion and formation of extra spores in diploids. Thus, Pcp1-dependent SPB quantity control is crucial for sexual reproduction and ploidy homeostasis in fission yeast.

Molecular and comparative genomic analyses reveal evolutionarily conserved and unique features of the Schizosaccharomyces japonicus mycelial growth and the underlying genomic changes.

Fungal pathogens, from phytopathogenic fungus to human pathogens, are able to alternate between the yeast-like form and filamentous forms. This morphological transition (dimorphism) is in close connection with their pathogenic lifestyles and with their responses to changing environmental conditions. The mechanisms governing these morphogenetic conversions are still not fully understood. Therefore, we studied the filamentous growth of the less-known, non-pathogenic dimorphic fission yeast, S. japonicus, which belongs to an ancient and early evolved branch of the Ascomycota. Its RNA sequencing revealed that several hundred genes were up- or down-regulated in the hyphae compared to the yeast-phase cells. These genes belonged to different GO categories, confirming that mycelial growth is a rather complex process. The genes of transport- and metabolic processes appeared especially in high numbers among them. High expression of genes involved in glycolysis and ethanol production was found in the hyphae, while other results pointed to the regulatory role of the protein kinase A (PKA) pathway. The homologues of 49 S. japonicus filament-associated genes were found by sequence alignments also in seven distantly related dimorphic and filamentous species. The comparative genomic analyses between S. japonicus and the closely related but non-dimorphic S. pombe shed some light on the differences in their genomes. All these data can contribute to a better understanding of hyphal growth and those genomic rearrangements that underlie it.

Loss of FZO1 gene results in changes of cell dynamics in fission yeast.

Mitochondrial fission and fusion dynamics are critical cellular processes, and abnormalities in these processes are associated with severe human disorders, such as Beckwith­Wiedemann syndrome, neurodegenerative diseases, Charcot­Marie­Tooth disease type 6, multiple symmetric lipomatosis and microcephaly. Fuzzy onions protein 1 (Fzo1p) regulates mitochondrial outer membrane fusion. In the present study, Schizosaccharomyces pombe (S. pombe) was used to explore the effect of FZO1 gene deletion on cell dynamics in mitosis. The mitochondrial morphology results showed that the mitochondria appeared to be fragmented and tubular in wild­type cells; however, they were observed to accumulate in fzo1Δ cells. The FZO1 gene deletion was demonstrated to result in slow proliferation, sporogenesis defects, increased microtubule (MT) number and actin contraction defects in S. pombe. The FZO1 gene deletion also affected the rate of spindle elongation and phase time at the metaphase and anaphase, as well as spindle MT organization. Live­cell imaging was performed on mutant strains to observe three distinct kinetochore behaviors (normal, lagging and mis­segregation), as well as abnormal spindle breakage. The FZO1 gene deletion resulted in coenzyme and intermediate metabolite abnormalities as determined via metabolomics analysis. It was concluded that the loss of FZO1 gene resulted in deficiencies in mitochondrial dynamics, which may result in deficiencies in spindle maintenance, chromosome segregation, spindle breakage, actin contraction, and coenzyme and intermediate metabolite levels.

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.

How Essential Kinesin-5 Becomes Non-Essential in Fission Yeast: Force Balance and Microtubule Dynamics Matter.

The bipolar mitotic spindle drives accurate chromosome segregation by capturing the kinetochore and pulling each set of sister chromatids to the opposite poles. In this review, we describe recent findings on the multiple pathways leading to bipolar spindle formation in fission yeast and discuss these results from a broader perspective. The roles of three mitotic kinesins (Kinesin-5, Kinesin-6 and Kinesin-14) in spindle assembly are depicted, and how a group of microtubule-associated proteins, sister chromatid cohesion and the kinetochore collaborate with these motors is shown. We have paid special attention to the molecular pathways that render otherwise essential Kinesin-5 to become non-essential: how cells build bipolar mitotic spindles without the need for Kinesin-5 and where the alternate forces come from are considered. We highlight the force balance for bipolar spindle assembly and explain how outward and inward forces are generated by various ways, in which the proper fine-tuning of microtubule dynamics plays a crucial role. Overall, these new pathways have illuminated the remarkable plasticity and adaptability of spindle mechanics. Kinesin molecules are regarded as prospective targets for cancer chemotherapy and many specific inhibitors have been developed. However, several hurdles have arisen against their clinical implementation. This review provides insight into possible strategies to overcome these challenges.

Myosins in Cytokinesis.

Nearly five decades of research have established myosin as the main motor responsible for cytokinesis in organisms on the branch of the phylogenetic tree that includes amoebas, fungi and animals. This research has grown to be more mechanistic over the past decade, so we now have computer simulations of physically reasonable models that explain how myosins contribute to the assembly and constriction of contractile rings that pinch dividing cells into two daughter cells. Isoforms of myosin-II, from the same family as muscle myosins, are the main myosins for cytokinesis, but other myosins contribute to cytokinesis in fission yeast. Progress has been made on how animal cells use Rho-GTPases to control the accumulation and activity of myosin-II at the site of cleavage, but the regulatory mechanisms are less clear in other systems.

Endocrine-Independent Cytotoxicity of Bisphenol A Is Mediated by Increased Levels of Reactive Oxygen Species and Affects Cell Cycle Progression.

Bisphenol A (BPA) is used for the production of plastics and epoxy resins, which are part of packaging materials for food and beverages, and can migrate into food and the environment, thus exposing human beings to its effects. Exposure to BPA has been associated with oxidative stress, cell cycle changes, and genotoxicity, and is mediated by its known endocrine-disrupting activity. Possible BPA cytotoxicity without mediation by estrogen receptors has been reported in the literature. Here, we show the toxic effects of BPA by live-cell imaging on the fission yeast Schizosaccharomyces pombe, an experimental model lacking estrogen receptors, which were in line with data from flow cytometry on intracellular oxidation (76.4 ± 14.4 and 19.4 ± 16.1% of fluorescent cells for BPA treatment and control, respectively; p < 0.05) as well as delay in cell cycle progression (after 90 min of experiment, 48.4 ± 4.30 and 64.6 ± 5.46% of cells with a 4C DNA content for BPA treatment and control, respectively; p < 0.05) upon exposure to BPA. These results strongly support the possibilities that BPA-induced cell cycle changes can be independent of estrogen receptors and that live-cell imaging is a powerful tool for genotoxic analysis.

Chemical screening identifies an extract from marine Pseudomonas sp.-PTR-08 as an anti-aging agent that promotes fission yeast longevity by modulating the Pap1-ctt1+ pathway and the cell cycle.

Aging is a degenerative process characterized by progressive deterioration of cellular components, ultimately resulting in mortality, in which massive accumulation of reactive oxygen species (ROS) and advanced glycation end products (AGEs) are implicated as crucial factors. At the same time, natural products are rich sources from which to isolate and characterize potential anti-aging compounds. The current study was designed to extract compounds from the marine bacterium Pseudomonas sp. and investigate their in vitro antioxidant and anti-glycation activities, as well as their in vivo effects on aging in the model organism Schizosaccharomyces pombe. In vitro assays showed that a Pseudomonas sp. PTR-08 extract exhibited the best antioxidant and anti-glycation activities. Further, direct administration of the extract significantly increased yeast longevity, accompanied by induction of the yeast oxidative stress response. Molecular analyses indicated that selected extract dramatically up-regulated the expression of pap1+, which encodes the transcriptional factor Pap1 and ctt1+, which encodes catalase, following H2O2 treatment. In line with these results, catalase activity significantly increased, leading to a decrease in intracellular ROS. In addition, this extract may delay the G1 phase of the yeast cell cycle, leading to an extended lifespan. Moreover, our findings indicated that the extract contains pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-, which substantially promotes anti-aging activity in yeast. However, further research must be conducted to better understand the role of this compound in our system.

Anchoring of actin to the plasma membrane enables tension production in the fission yeast cytokinetic ring.

The cytokinetic ring generates tensile force that drives cell division, but how tension emerges from the relatively disordered ring organization remains unclear. Long ago, a musclelike sliding filament mechanism was proposed, but evidence for sarcomeric order is lacking. Here we present quantitative evidence that in fission yeast, ring tension originates from barbed-end anchoring of actin filaments to the plasma membrane, providing resistance to myosin forces that enables filaments to develop tension. The role of anchoring was highlighted by experiments on isolated fission yeast rings, where sections of ring became unanchored from the membrane and shortened ∼30-fold faster than normal. The dramatically elevated constriction rates are unexplained. Here we present a molecularly explicit simulation of constricting partially anchored rings as studied in these experiments. Simulations accurately reproduced the experimental constriction rates and showed that following anchor release, a segment becomes tensionless and shortens via a novel noncontractile reeling-in mechanism at about the velocity of load-free myosin II. The ends are reeled in by barbed end-anchored actin filaments in adjacent segments. Other actin anchoring schemes failed to constrict rings. Our results quantitatively support a specific organization and anchoring scheme that generate tension in the cytokinetic ring.

Spindle pole body movement is affected by glucose and ammonium chloride in fission yeast.

The complexity of chromatin dynamics is orchestrated by several active processes. In fission yeast, the centromeres are clustered around the spindle pole body (SPB) and oscillate in a microtubule- and adenosine triphosphate (ATP)-dependent manner. However, whether and how SPB oscillation are affected by different environmental conditions remain poorly understood. In this study, we quantitated movements of the SPB component, which colocalizes with the centromere in fission yeast. We found that SPB movement was significantly reduced at low glucose concentrations. Movement of the SPB was also affected by the presence of ammonium chloride. Power spectral analysis revealed that periodic movement of the SPB is disrupted by low glucose concentrations. Measurement of ATP levels in living cells by quantitative single-cell imaging suggests that ATP levels are not the only determinant of SPB movement. Our results provide novel insight into how SPB movement is regulated by cellular energy status and additional factors such as the medium nutritional composition.

Casein kinase II-dependent phosphorylation of DNA topoisomerase II suppresses the effect of a catalytic topo II inhibitor, ICRF-193, in fission yeast.

DNA topoisomerase II (topo II) regulates the topological state of DNA and is necessary for DNA replication, transcription, and chromosome segregation. Topo II has essential functions in cell proliferation and therefore is a critical target of anticancer drugs. In this study, using Phos-tag SDS-PAGE analysis in fission yeast (Schizosaccharomyces pombe), we identified casein kinase II (Cka1/CKII)-dependent phosphorylation at the C-terminal residues Ser1363 and Ser1364 in topo II. We found that this phosphorylation decreases the inhibitory effect of an anticancer catalytic inhibitor of topo II, ICRF-193, on mitosis. Consistent with the constitutive activity of Cka1/CKII, Ser1363 and Ser1364 phosphorylation of topo II was stably maintained throughout the cell cycle. We demonstrate that ICRF-193-induced chromosomal mis-segregation is further exacerbated in two temperature-sensitive mutants, cka1-372 and cka1/orb5-19, of the catalytic subunit of CKII or in the topo II nonphosphorylatable alanine double mutant top2-S1363A,S1364A but not in cells of the phosphomimetic glutamate double mutant top2-S1363E,S1364E Our results suggest that Ser1363 and Ser1364 in topo II are targeted by Cka1/CKII kinase and that their phosphorylation facilitates topo II ATPase activity in the N-terminal region, which regulates protein turnover on chromosome DNA. Because CKII-mediated phosphorylation of the topo II C-terminal domain appears to be evolutionarily conserved, including in humans, we propose that attenuation of CKII-controlled topo II phosphorylation along with catalytic topo II inhibition may promote anticancer effects.

The fission yeast Greatwall-Endosulfine pathway is required for proper quiescence/G0 phase entry and maintenance.

Cell proliferation and cellular quiescence/G0 phase must be regulated in response to intra-/extracellular environments, and such regulation is achieved by the orchestration of protein kinases and protein phosphatases. Here, we investigated fission yeast potential orthologs (Cek1, Ppk18 and Ppk31) of the metazoan Greatwall kinase (Gwl), which inhibits type-2A protein phosphatase with B55 subunit (PP2AB55 ) by phosphorylating and activating the PP2AB55 inhibitors, α-endosulfine/ARPP-19 (Ensa/ARPP-19). Gwl and Ensa/ARPP-19 regulate mitosis; however, we found Ppk18, Cek1 and Mug134/Igo1, the counterpart of Ensa/ARPP-19, are not essential for normal mitosis but regulate nitrogen starvation (-N)-induced proper G0 entry and maintenance. Genetic and biochemical analyses indicated that the conserved Gwl site (serine 64) was phosphorylated in the G0 phase in a Ppk18-dependent manner, and the phosphorylated Mug134/Igo1 inhibited PP2AB55 in vitro. The alanine substitution of the serine 64 caused defects in G0 entry and maintenance as well as the mug134/igo1+ deletion. These results indicate that PP2AB55 activity must be regulated properly to establish the G0 phase. Consistently, simultaneous deletion of the B55 gene with mug134/igo1+ partially rescued the Mug134/Igo1 mutant phenotype. We suggest that in fission yeast, PP2AB55 regulation by the Ppk18-Mug134/Igo1 pathway is required for G0 entry and establishment of robust viability during the G0 phase.

Implications of alternative routes to APC/C inhibition by the mitotic checkpoint complex.

The mitotic checkpoint (also called spindle assembly checkpoint) is a signaling pathway that ensures faithful chromosome segregation. Mitotic checkpoint proteins inhibit the anaphase-promoting complex (APC/C) and its activator Cdc20 to prevent precocious anaphase. Checkpoint signaling leads to a complex of APC/C, Cdc20, and checkpoint proteins, in which the APC/C is inactive. In principle, this final product of the mitotic checkpoint can be obtained via different pathways, whose relevance still needs to be fully ascertained experimentally. Here, we use mathematical models to compare the implications on checkpoint response of the possible pathways leading to APC/C inhibition. We identify a previously unrecognized funneling effect for Cdc20, which favors Cdc20 incorporation into the inhibitory complex and therefore promotes checkpoint activity. Furthermore, we find that the presence or absence of one specific assembly reaction determines whether the checkpoint remains functional at elevated levels of Cdc20, which can occur in cancer cells. Our results reveal the inhibitory logics behind checkpoint activity, predict checkpoint efficiency in perturbed situations, and could inform molecular strategies to treat malignancies that exhibit Cdc20 overexpression.

Fission yeast Adf1 is necessary for reassembly of actin filaments into the contractile ring during cytokinesis.

ADF/cofilin family proteins quickly disassemble actin in vitro, and are thought to be involved in various actin dynamics in the cell. Adf1 is a member of this family proteins expressed in fission yeast, and is thought to play roles in actin patch dynamics and also contractile ring formation during cytokinesis. We aimed to understand the function of this protein in cytokinesis in detail using the temperature-sensitive mutant adf1-1. Adf1 inactivation at a restrictive temperature during late G2 phase led to a clustering of actin patches at the cell ends. It was apparent that the inactivation occurred only in a few minutes. Furthermore, we found that the actin clusters migrated to the division site during anaphase possibly by the function of both myosin 5-1 and a myosin II. The migrated actin clusters, however, were not organized into the contractile ring. When Adf1 was inactivated at mid-anaphase B before contractile ring assembly, the ring was not formed, but it was formed when Adf1 was inactivated after this point. We conclude that Adf1 functions in the interphase actin dynamics and formation of the contractile ring during mitosis.

Genome-wide evidences of bisphenol a toxicity using Schizosaccharomyces pombe.

To clarify reliable toxic mechanisms of bisphenol A (BPA), an endocrine disrupting chemical, we approached an alternative animal and whole genome analyses with the yeast knockout library (YKO) of Schizosaccharomyces pombe. As results, the 50% growth inhibition concentrations (GI50) of BPA was approximately 600 µM and the YKO-three step screening revealed the top 10 target candidate genes including dbp2, utp18, srs1, tif224, use1, qcr1, etc. The screening results were confirmed in human embryonic stem cell (hES)-derived hepatic cells and HepG2 human liver cancer cells. We found BPA down-regulated UQCRC, the human orthlog of S. pombe- qcr1, as a part of the mitochondrial respiratory chain, in HepG2 cells and hESs during cell differentiation into hepatic cells. Therefore, BPA may induce mitochondrial dysfunction and disruption of differentiation by suppressing UQCRC1.

Gamete fusion triggers bipartite transcription factor assembly to block re-fertilization.

The ploidy cycle, which is integral to sexual reproduction, requires meiosis to halve chromosome numbers as well as mechanisms that ensure zygotes are formed by exactly two partners1-4. During sexual reproduction of the fungal model organism Schizosaccharomyces pombe, haploid P and M cells fuse to form a diploid zygote that immediately enters meiosis5. Here we reveal that rapid post-fusion reconstitution of a bipartite transcription factor blocks re-fertilization. We first identify mutants that undergo transient cell fusion involving cytosol exchange but not karyogamy, and show that this drives distinct cell fates in the two gametes. The P partner undergoes lethal haploid meiosis, whereas the M cell persists in mating. The zygotic transcription that drives meiosis is rapidly initiated first from the P parental genome, even in wild-type cells. This asymmetric gene expression depends on a bipartite complex formed post-fusion between the cytosolic M-cell-specific peptide Mi and the nuclear P-cell-specific homeobox protein Pi6,7, which captures Mi in the P nucleus. Zygotic transcription is thus poised to initiate in the P nucleus as fast as Mi reaches it after fusion, a design that we reconstruct using two synthetic interactors localized to the nucleus and the cytosol of two respective partner cells. Notably, delaying zygotic transcription-by postponing Mi expression or deleting its transcriptional target in the P genome-leads to zygotes fusing with additional gametes, thus forming polyploids and eventually aneuploid progeny. The signalling cascade to block re-fertilization shares components with, but bifurcates from, meiotic induction8-10. Thus, a cytoplasmic connection upon gamete fusion leads to asymmetric reconstitution of a bipartite transcription factor to rapidly block re-fertilization and induce meiosis, ensuring genome maintenance during sexual reproduction.

Discovery of Presaccharothriolide X, a Retro-Michael Reaction Product of Saccharothriolide B, from the Rare Actinomycete Saccharothrix sp. A1506.

The highly reactive precursor molecule, presaccharothriolide X, was successfully isolated from the rare actinomycete Saccharothrix sp. A1506. The comparable biological activity of presaccharothriolide X and its Michael addition product saccharothriolide B unveils a unique masking/activating property of 2-aminophenol. Unexpectedly, 2-aminophenol in saccharothriolide B was eliminated through a retro-Michael reaction, to yield presaccharothriolide X under physiological conditions. 2-Aminophenol might be developed into a useful protecting group for bioactive small molecules with an α,ß-unsaturated ketone.

Dependency relationships within the fission yeast polarity network.

The ability to regulate polarised cell growth is crucial to maintain the viability of cells. Growth is modulated to facilitate essential cell functions and respond to the external environment. Failure to do so can lead to numerous developmental and disease states, including cancer. We have undertaken a detailed analysis of the regulatory interplay between molecules involved in the regulation and maintenance of polarised cell growth within fission yeast. Internally controlled live cell imaging was used to examine interactions between 10 key polarity proteins. Analysis reveals interplay between the microtubule and actin cytoskeletons, as well as multiple novel dependency pathways and feedback networks between groups of proteins. This study provides important insights into the conserved regulation of polarised cell growth within eukaryotes.

Parameter uncertainty quantification using surrogate models applied to a spatial model of yeast mating polarization.

A common challenge in systems biology is quantifying the effects of unknown parameters and estimating parameter values from data. For many systems, this task is computationally intractable due to expensive model evaluations and large numbers of parameters. In this work, we investigate a new method for performing sensitivity analysis and parameter estimation of complex biological models using techniques from uncertainty quantification. The primary advance is a significant improvement in computational efficiency from the replacement of model simulation by evaluation of a polynomial surrogate model. We demonstrate the method on two models of mating in budding yeast: a smaller ODE model of the heterotrimeric G-protein cycle, and a larger spatial model of pheromone-induced cell polarization. A small number of model simulations are used to fit the polynomial surrogates, which are then used to calculate global parameter sensitivities. The surrogate models also allow rapid Bayesian inference of the parameters via Markov chain Monte Carlo (MCMC) by eliminating model simulations at each step. Application to the ODE model shows results consistent with published single-point estimates for the model and data, with the added benefit of calculating the correlations between pairs of parameters. On the larger PDE model, the surrogate models allowed convergence for the distribution of 15 parameters, which otherwise would have been computationally prohibitive using simulations at each MCMC step. We inferred parameter distributions that in certain cases peaked at values different from published values, and showed that a wide range of parameters would permit polarization in the model. Strikingly our results suggested different diffusion constants for active versus inactive Cdc42 to achieve good polarization, which is consistent with experimental observations in another yeast species S. pombe.