Programmed cell death in fission yeast Schizosaccharomyces pombe

Yeasts have proven to be invaluable, genetically tractable systems to study various fundamental biological processes including programmed cell death. Recent advances in the elucidation of the molecular pathways underlying apoptotic cell death in yeasts have revealed remarkable similarities to mammalian apoptosis at cellular, organelle and macromolecular levels, thus making a strong case for the relevance of yeast models of regulated cell death. Programmed cell death has been reported in fission yeast Schizosaccharomyces pombe, primarily in the contexts of perturbed intracellular lipid metabolism, defective DNA replication, improper mitotic entry, chronological and replicative aging. Here we review the current understanding of the programmed cell death in fission yeast, paying particular attention to lipid-induced cell death. We discuss our recent findings that fission yeast exhibits plasticity of apoptotic and non-apoptotic modes of cell death in response to different lipid stimuli and growth conditions, and that mitochondria, reactive oxygen species and novel cell death mediators including metacaspase Pca1, SpRad9 and Pck1 are involved in the lipotoxic cell death. We also present perspectives on how various aspects of the cell and molecular biology of this organism can be explored to shed light on the governing principles underlying lipid-mediated signaling and cell demise.     

Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a ‘module’, can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism.     

DNA repair in the fission yeast, Schizosaccharomyces pombe

Mutants of the fission yeast Schizosaccharomyces pombe which are sensitive to UV and/or γ-irradiation have been assigned to 23 complementation groups, which can be assigned to three phenotypic groups. We have cloned genes which correct the deficiency in mutants corresponding to 12 of the complementation groups. Three genes in the excision-repair pathway have a high degree of sequence conservation with excision-repair genes from the evolutionarily distant budding yeast Saccharomyces cerevisiae. In contrast, those genes in the recombination repair pathway which have been characterised so far, show little homology with any previously characterised genes.     

Sexual co-flocculation by heterothallic cells of the fission yeast Schizosaccharomyces pombe modulated by medium constituents

Novel simple synthetic media for inducing sexual co-flocculation in a short time after mixing heterothallic fission-yeast (Schizosaccharomyces pombe) cells of h^- and h⁺ were devised; The most effective of these, mannose synthetic medium (MSM), contains 0.4% mannose as a carbon source in addition to galactose, KH₂PO₄ (pH4.0) and 4 vitamins. The addition of galactose to the medium suppressed the asexual self-flocculation but rather promoted the sexual co-flocculation. By transferring and mixing h^- and h⁺ cells grown in malt-extract broth plus galactose into MSM, these heterothallic strains were revealed to be sexually ready through a long period of the log to stationary phases. Furthermore, a variety of C sources and NH₄Cl at various concentrations in various media were examined for their effects upon sexual co-flocculation, conjugation and sporulation; it was found that the sugar concentration strictly affected the progress of the sequence of sexual reproduction at 26°C but not 30°C and that sexual co-flocculation of the heterothallic strains was induced only under lower concentrations of C and N source than that for the homothallic one.     

Generation of a set of conditional analog-sensitive alleles of essential protein kinases in the fission yeast Schizosaccharomyces pombe

The genome of the fission yeast Schizosaccharomyces pombe encodes for 17 protein kinases that are essential for viability. Studies of the essential kinases often require the use of mutant strains carrying conditional alleles. To inactivate these kinases conditionally, we applied a recently developed chemical genetic strategy. The mutation of a single residue in the ATP-binding pocket confers sensitivity to small-molecule inhibitors, allowing for specific inactivation of the modified kinase. Using this approach, we constructed conditional analog-sensitive alleles of 13 essential protein kinases in the fission yeast S. pombe.     

The proteolytic system of the fission yeast Schizosaccharomyces pombe

Proteinase and peptidase activities of the fission yeast Schizosaccharomyces pombe were investigated. Several intracellular proteolytic enzymes were found: two endoproteinases, one carboxypeptidase, one aminopeptidase and one dipeptidyl-aminopeptidase. In addition, proteinase inhibitors were detected. In fresh crude extracts an activation procedure is needed to measure maximal activities of endoproteinases and carboxypeptidase, whose level is markedly dependent on growth medium composition and on growth phase, while aminopeptidase and dipeptidyl-aminopeptidase activities are very little, if at all, regulated by the carbon source.     

Pom1 and cell size homeostasis in fission yeast

Cells sense their size and use this information to coordinate cell division with cell growth to maintain a constant cell size within a given population. A model has been proposed for cell size control in the rod-shaped cells of the fission yeast, Schizosaccharomyces pombe. This involves a protein localized to the cell ends, which inhibits mitotic activators in the middle of the cell in a cell size-dependent manner. This protein, Pom1, along with another tip-localized protein, Nif1, have been implicated as direct sensors of cell size controlling the onset of mitosis. Here we have investigated cell size variability and size homeostasis at the G₂/M transition, focusing on the role of pom1 and nif1. Cells deleted for either of these 2 genes show wild-type size homeostasis both in size variability analyses and size homeostasis experiments. This indicates that these genes do not have a critical role as direct cell size sensors in the control mechanism. Cell size homeostasis also seems to be independent of Cdc2–Tyr15 phosphorylation, suggesting that the size sensing mechanism in fission yeast may act through an unidentified pathway regulating CDK activity by an unknown mechanism.     

HIV-1 Vpr-induced cell death in Schizosaccharomyces pombe is reminiscent of apoptosis

Human immunodeficiency virus type 1 （HIV-1） Vpr induces cell death in mammalian and fssion yeast cells, suggesting that Vpr may affect a conserved cellular process. It is unclear, however, whether Vpr-induced yeast cell death mimics Vpr-mediated apoptosis in mammalian cells. We have recently identified a number of Vpr suppressors that not only suppress Vpr-induced cell death in fission yeast, but also block Vpr-induced apoptosis in mammalian cells. These findings suggest that Vpr-induced cell death in yeast may resemble some of the apoptotic processes of mammalian cells. The goal of this study was to develop and validate a fission yeast model system for future studies of apoptosis. Similar to Vpr-induced apoptosis in mammalian cells, we show here that Vpr in fission yeast promotes phosphatidylserine externalization and induces hyperpolarization of mitochondria, leading to changes of mitochondrial membrane potential. Moreover, Vpr triggers production of reactive oxygen species （ROS）, indicating that the apoptotic-like cell death might be mediated by ROS. Interestingly, Vpr induces unique morphologic changes in mitochondria that may provide a simple marker for measuring the apoptotic-like process in fission yeast. To verify this possibility, we tested two Vpr suppressors （EF2 and Hspl6） that suppress Vpr-induced apoptosis in mammalian cells in addition to a newly identified Vpr suppressor （Skpl）. All three proteins abolished cell death mediated by Vpr and restored normal mitochondrial morphology in the yeast cells. In conclusion, Vpr-induced cell death in fission yeast resembles the mammalian apoptotic process. Fission yeast may thus potentially be used as a simple model organism for the future study of the apoptotic-like process induced by Vpr and other proapoptotic agents.     

A knockout screen for protein kinases required for the proper meiotic segregation of chromosomes in the fission yeast Schizosaccharomyces pombe

The reduction of chromosome number during meiosis is achieved by two successive rounds of chromosome segregation after just single round of DNA replication. To identify novel proteins required for the proper segregation of chromosomes during meiosis, we analyzed the consequences of deleting Schizosaccharomyces pombe genes predicted to encode protein kinases that are not essential for cell viability. We show that Mph1, a member of the Mps1 family of spindle assembly checkpoint kinases, is required to prevent meiosis I homolog non-disjunction. We also provide evidence for a novel function of Spo4, the fission yeast ortholog of Dbf4-dependent Cdc7 kinase, in regulating the length of anaphase II spindles. In the absence of Spo4, abnormally elongated anaphase II spindles frequently overlap and thus destroy the linear order of nuclei in the ascus. Our observation that the spo4Δ mutant phenotype can be partially suppressed by inhibiting Cdc2-as suggests that dysregulation of the activity of this cyclin-dependent kinase may cause abnormal elongation of anaphase II spindles in spo4Δ mutant cells.     

Analysis of stress granule assembly in Schizosaccharomyces pombe

Stress granules (SGs) are cytoplasmic aggregates of RNA and proteins in eukaryotic cells that are rapidly induced in response to environmental stress, but are not seen in cells growing under favorable conditions. SGs have been primarily studied in mammalian cells. The existence of SGs in the fission yeast and the distantly related budding yeast was demonstrated only recently. In both species, they contain many orthologs of the proteins seen in mammalian SGs. In this study, we have characterized these proteins and determined their involvement in the assembly of fission yeast SGs, in particular, the homolog of human G3BP proteins. G3BP interacts with the deubiquitinating protease USP10 and plays an important role in the assembly of SGs. We have also identified Ubp3, an ortholog of USP10, as an interaction partner of the fission yeast G3BP-like protein Nxt3 and required for its stability. Under thermal stress, like their human orthologs, both Nxt3 and Ubp3 rapidly relocalize to cytoplasmic foci that contain the SG marker poly(A)-binding protein Pabp. However, in contrast to G3BP1 and USP10, neither deletion nor overexpression of nxt3(+) or ubp3(+) affected the assembly of fission yeast SGs as judged by the relocalization of Pabp. Similar results were observed in mutants defective in orthologs of SG components that are known to affect SG assembly in human and in budding yeast, such as ataxia-2 and TIA-like proteins. Together, our data indicate that despite similar protein compositions, the underlying molecular mechanisms for the assembly of SGs could be distinct between species.     

Dikaryotic cell division of the fission yeast Schizosaccharomyces pombe

Dikaryons, cells with two haploid nuclei contributed by the members of a mating pair, are part of the life cycle of many filamentous fungi, but the molecular mechanisms underlying the division of dikaryons are largely unknown. We found that the fission yeast Schizosaccharomyces pombe has a latent ability to divide as a dikaryon. Cells capable of restarting the mitotic cycle with two nuclei were prepared by transient inactivation of the septation initiation network. Close pairing of the two nuclei before mitosis was dependent on minus-end-directed kinesin Klp2p and was essential for propagation as a dikaryon. The two spindles extended in opposite directions, keeping their old spindle pole bodies at the prospective site of cell division until the mid-anaphase. The spindles then overlapped, exchanging the inner nuclei. Finally, twin mitosis was followed by a single cytokinesis, producing two daughter dikaryons carrying copies of the original pair of nuclei.     

Urea enhances cell lysis of Schizosaccharomyces pombe ura4 mutants

Cell lysis is induced in Schizosaccharomyces pombe ?ura4 cells grown in YPD medium, which contains yeast extract, polypeptone, and glucose. To identify the medium components that induce cell lysis, we first tested various kinds of yeast extracts from different suppliers. Cell lysis of ?ura4 cells on YE medium was observed when yeast extracts from OXOID, BD, Oriental, and Difco were used, but not when using yeast extract from Kyokuto. To determine which compounds induced cell lysis, we subjected yeast extract and polypeptone to GC-MS analysis. Ten kinds of compounds were detected in OXOID and BD yeast extracts, but not in Kyokuto yeast extract. Among them was urea, which was also present in polypeptone, and it clearly induced cell lysis. Deletion of the ure2 gene, which is responsible for utilizing urea, abolished the lytic effect of urea. The effect of urea was suppressed by deletion of pub1, and a similar phenotype was observed in the presence of polypeptone. Thus, urea is an inducer of cell lysis in S. pombe ?ura4 cells.     

Role for trehalase during germination of spores in the fission yeast Schizosaccharomyces pombe

Spores from Schizosaccharomyces pombe contain neutral and acid trehalases. When spores from strains disrupted for ntp1(+), which encodes neutral trehalase, were induced to germinate, the onset of the process was markedly delayed as compared to wild-type spores. Further outgrowth was also reduced. Dormant spores lacking neutral trehalase contained twice the amount of trehalose present in wild-type spores and mobilised the intracellular pool of trehalose at a slower rate during germination. Inhibition by phloridzin of the sporulation-specific acid trehalase in ntp1-disrupted spores arrested germination completely while prompting no effect on wild-type spores. These results suggest that the two trehalase enzymes may support the utilisation of trehalose during germination but neutral trehalase is required for a more rapid and efficient process.     

Modelling the fission yeast cell cycle.

The molecular networks regulating basic physiological processes in a cell can be converted into mathematical equations (eg differential equations) and solved by a computer. The division cycle of eukaryotic cells is an important example of such a control system, and fission yeast is an excellent test organism for the computational modelling approach. The mathematical model is tested by simulating wild-type cells and many known cell cycle mutants. This paper describes an example where this approach is useful in understanding multiple rounds of DNA synthesis (endoreplication) in fission yeast cells that lack the main (B-type) mitotic cyclin, Cdc13. It is proposed that the key physiological variable driving progression through the cell cycle during balanced growth and division is the mass/DNA ratio, rather than the mass/nucleus ratio.     

Effects of pressure stress on the fission yeast Schizosaccharomyces pombe cold-sensitive mutant nda3

To investigate the influence of pressure stress on the cell cycle of Schizosaccharomyces pombe, we used a cold-sensitive nda3-KM311 mutant which arrests cell division at a step similar to the mitotic prophase, proposed by Hiraoka and colleagues (Cell 39 (1984) 349-358), under the restrictive temperature, 20 degrees C. The nda3-KM311 cells were first aerobically grown at 30 degrees C, transferred to 20 degrees C for 4 h and shifted to a permissive temperature of 36 degrees C for 15 min. The cells were treated with 100-200 MPa pressure and studied by electron and fluorescence microscopy. At 100 MPa, the nuclear membrane was damaged and the matrix of mitochondria had an electron-dense area. At 150 MPa, the nuclear membrane was broken over broad areas; numerous small vacuoles had fused into large pieces. Actin patches were concentrated in the central region and actin rings were seen in the 20 degrees C-grown cells. Even at 100 MPa, specific actin distribution was lost. Although at 100 MPa, long and fine actin cables were seen all over the cells, large actin patches and the actin rings remained in the center of the cell. They changed into thick and short cables at 150 MPa and above 200 MPa they decomposed but the actin ring was visible even with faint fluorescence. Immunoelectron microscopic observation confirmed this phenomenon.     

Programmed cell death in fission yeast

Recently a metacaspase, encoded by YCA1, has been implicated in a primitive form of apoptosis or programmed cell death in yeast. Previously it had been shown that over-expression of mammalian pro-apoptotic proteins can induce cell death in yeast, but the mechanism of how cell death occurred was not clearly established. More recently, it has been shown that DNA or oxidative damage, or other cell cycle blocks, can result in cell death that mimics apoptosis in higher cells. Also, in fission yeast deletion of genes required for triacylglycerol synthesis leads to cell death and expression of apoptotic markers. A metacaspase sharing greater than 40% identity to budding yeast Yca1 has been identified in fission yeast, however, its role in programmed cell death is not yet known. Analysis of the genetic pathways that influence cell death in yeast may provide insights into the mechanisms of apoptosis in all eukaryotic organisms.