Computational modeling of chromosome re-replication in mutant strains of fission yeast

Typically cells replicate their genome only once per division cycle, but under some circumstances, both natural and unnatural, cells synthesize an overabundance of DNA, either in a disorganized manner (“overreplication”) or by a systematic doubling of chromosome number (“endoreplication”). These variations on the theme of DNA replication and division have been studied in strains of fission yeast, Schizosaccharomyces pombe, carrying mutations that interfere with the function of mitotic cyclin-dependent kinase (Cdk1:Cdc13) without impeding the roles of DNA-replication loading factor (Cdc18) and S-phase cyclin-dependent kinase (Cdk1:Cig2). Some of these mutations support endoreplication, and some overreplication. In this paper, we propose a dynamical model of the interactions among the proteins governing DNA replication and cell division in fission yeast. By computational simulations of the mathematical model, we account for the observed phenotypes of these re-replicating mutants, and by theoretical analysis of the dynamical system, we provide insight into the molecular distinctions between overreplicating and endoreplicating cells. In the case of induced overproduction of regulatory proteins, our model predicts that cells first switch from normal mitotic cell cycles to growth-controlled endoreplication, and ultimately to disorganized overreplication, parallel to the slow increase of protein to very high levels.     

Comparison of a coq7 deletion mutant with other respiration-defective mutants in fission yeast

Among the steps in ubiquinone biosynthesis, that catalyzed by the product of the clk-1/coq7 gene has received considerable attention because of its relevance to life span in Caenorhabditis elegans. We analyzed the coq7 ortholog (denoted coq7) in Schizosaccharomyces pombe, to determine whether coq7 has specific roles that differ from those of other coq genes. We first confirmed that coq7 is necessary for the penultimate step in ubiquinone biosynthesis, from the observation that the deletion mutant accumulated the ubiquinone precursor demethoxyubiquinone-10 instead of ubiquinone-10. The coq7 mutant displayed phenotypes characteristic of other ubiquinone-deficient Sc. pombe mutants, namely, hypersensitivity to hydrogen peroxide, a requirement for antioxidants for growth on minimal medium, and an elevated production of sulfide. To compare these phenotypes with those of other respiration-deficient mutants, we constructed cytochrome c (cyc1) and coq3 deletion mutants. We also assessed accumulation of oxidative stress in various ubiquinone-deficient strains and in the cyc1 mutant by measuring mRNA levels of stress-inducible genes and the phosphorylation level of the Spc1 MAP kinase. Induction of ctt1, encoding catalase, and apt1, encoding a 25 kDa protein, but not that of gpx1, encoding glutathione peroxidase, was indistinguishable in four ubiquinone-deficient mutants, indicating that the oxidative stress response operates at similar levels in the tested strains. One new phenotype was observed, namely, loss of viability in stationary phase (chronological life span) in both the ubiquinone-deficient mutant and in the cyc1 mutant. Finally, Coq7 was found to localize in mitochondria, consistent with the possibility that ubiquinone biosynthesis occurs in mitochondria in yeasts. In summary, our results indicate that coq7 is required for ubiquinone biosynthesis and the coq7 mutant is not distinguishable from other ubiquinone-deficient mutants, except that its phenotypes are more pronounced than those of the cyc1 mutant.     

Cell phenotypes of a mutant in the gene encoding a Rad51 paralog in fission yeast

The discovery of three Rad51 paralogs in Saccharomyces cerevisiae (Rad55, Rad57, and Dmc1), four in Schizosaccharomyces pombe (Rhp55, Rhp57, Rlp1, and Dmc1), and six in human (Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3, and Dmc1) indicate the functional diversity and specialization of RecA-like proteins in the line from the lower to higher organisms. This paper reports characterization of a number of mitotic and meiotic phenotypes of the cells mutant in rlp1 gene, encoding a paralog of Rad51, in fission yeasts. No evident role of Rlp1 protein in the repair of spontaneous lesions emerging during mating type switching was found. Rlp1 does not interact physically with Dmc1. An elevated expression of rhp51 has a dominant negative effect on the cell survivability of rlp1Δ mutant exposed to a DNA-damaging agent. We assume that Rlp1 acts at the stages of recombination connected with disassembling of the nucleoprotein filament formed by Rhp51 protein.     

Cell phenotypes of a mutant in the gene encoding a Rad51 paralog in fission yeast

The discovery of three Rad51 paralogs in Saccharomyces cerevisiae (Rad55, Rad57, and Dmc1), four in Schizosaccharomyces pombe (Rhp55, Rhp57, Rlp 1, and Dmc 1), and six in human (Rad51 B, Rad51 C, Rad51 D, Xrcc2, Xrcc3, and Dmcl) indicate the functional diversity and specialization of RecA-like proteins in the line from the lower to higher organisms. This paper reports characterization of a number of mitotic and meiotic phenotypes of the cells mutant in rlpl gene, encoding a paralog of Rad5 1, in fission yeasts. No evident role of Rlp I protein in the repair of spontaneous lesions emerging during mating type switching was found. Rlpl does not interact physically with Dmcl. An elevated expression of rhp51 has a dominant negative effect on the cell survivability of rlpl mutant exposed to a DNA-damaging agent. We assume that Rlp 1 acts at the stages of recombination connected with disassembling of the nucleoprotein filament formed by Rhp51 protein.     

A wat1 mutant of fission yeast is defective in cell morphology

The organization of the actin cytoskeleton plays an integral role in cell morphogenesis of all eukaryotes. We have isolated a temperature-sensitive mutant in Schizosaccharomyces pombe, wat1-1, in which acting patches are delocalized, resulting in an elliptically shaped cell phenotype. Molecular cloning and DNA sequencing of wat1 ⁺ showed that the gene encodes a 314 residue protein containing WD-40 repeats. Cells lacking wat1 ⁺ are slow growing but viable at 25°?C and temperature-sensitive for growth above 33°?C. At restrictive temperature, wat1-d strains are phenotypically indistinguishable from wat1-1. When combined with a deletion for the wat1 ⁺ gene, cdc mutants failed to elongate at restrictive temperature and exhibited alterations in actin patch localization. This analysis suggests that wat1 ⁺ is required directly or indirectly for polarized cell growth in S. pombe. Wat1p and a functional, epitope-tagged, version of Wat1p can be overproduced without inducing alterations in cell morphology.     

Uncontrolled septation in a cell division cycle mutant of the fission yeast Schizosaccharomyces pombe.

A temperature-sensitive Schizosaccharomyces pombe mutant, cdc16-116, has been isolated which undergoes uncontrolled septation during its cell division cycle. The mutant accumulates two types of cells after 3 h of growth at the restrictive temperature: (i) type I cells (85% of the population), which complete nuclear division and then form up to five septa between the divided nuclei; and (ii) type II cells (15% of the population), which form an asymmetrically situated septum in the absence of any nuclear division. cdc16-116 is a monogenic recessive mutation unlinked to any previously known cdc gene of S. pombe. It is not affected in a previously reported control by which septation is dependent upon completion of nuclear division. We propose the cdc16-116 is unable to complete septum formation and proceed to cell separation and is also defective in a control which prevents the manufacture of more than one septum in each cell cycle.     

Oxygen toxicity in a fission yeast

Continuous exposure of synchronous cultures of Schizosaccharomyces pombe to 2.0 atmospheres oxygen beginning at any point in the first two-thirds of the cell cycle prevented subsequent cell division. Similar exposure during the last one-third of the cell cycle did not prevent cell division. The inhibition of division was totally reversible. Exposure to 2.0 atmospheres oxygen for 2.5 hours did not affect oxygen consumption. Oxygen at 1.0 atmospheres reduced growth rate and protein synthesis by 44%. Similar exposure to 1.0 atmospheres reduced transport of glycine-¹⁴C, L-leucine-¹⁴C, and uracil-¹⁴C by 95%, 73%, and 89% respectively. Analysis of the kinetics of uptake of these materials showed noncompetitive inhibition of transport by oxygen. The primary effect in rapidly appearing oxygen toxicity apparently involved interference with the transport capabilities of the cell membrane.     

Characterization of a fission yeast mutant which displays defects in cell wall integrity and cytokinesis.

The fission yeast cps6-153 mutant was originally isolated based on its hypersensitivity to the spindle poison isopropyl N-3-chlorophenyl carbamate (CIPC). The mutant also shows defects in both cell wall integrity and cytokinesis, resulting in the accumulation of unseparated cells with weakened cell walls. The arrested cells display a disoriented alignment of cytoplasmic microtubules. When the mutant cells are cultivated at high temperature (35 degrees C), both cell walls and septa become very thick. Electron microscopy revealed the disorganized structure of the thickened cell walls and septa, in which fibrillar components were not completely masked with an amorphous matrix. rad25+ was cloned from a genomic library by complementation of the mutant phenotypes, suggesting the involvement of Rad25p, one of two 14-3-3 proteins in S. pombe, in the pathway of cell wall integrity and cytokinesis.     

In vitro reactivation of spindle elongation in fission yeast nuc2 mutant cells

To investigate the mechanisms of spindle elongation and chromosome separation in the fission yeast Schizosaccharomyces pombe, we have developed an in vitro assay using a temperature-sensitive mutant strain, nuc2. At the restrictive temperature, nuc2 cells are arrested at a metaphase-like stage with short spindles and condensed chromosomes. After permeabilization of spheroplasts of the arrested cells, spindle elongation was reactivated by addition of ATP and neurotubulin both at the restrictive and the permissive temperatures, but chromosome separation was not. This suggests that the nuc2 cells are impaired in function at a stage before sister chromatid disjunction. Spindle elongation required both ATP and exogenous tubulin and was inhibited by adenylyl imidodiphosphate (AMPPNP) or vanadate. The ends of yeast half-spindle microtubules pulse-labeled with biotinylated tubulin moved past each other during spindle elongation and a gap formed between the original half-spindles. These results suggest that the primary mechanochemical event responsible for spindle elongation is the sliding apart of antiparallel microtubules of the two half-spindles.     

Genetic and molecular characterization of Skb15, a highly conserved inhibitor of the fission yeast PAK, Shk1

The p21-activated kinase, Shk1, is essential for viability, establishment and maintenance of cell polarity, and proper mating response in the fission yeast, Schizosaccharomyces pombe. Here we describe the characterization of a highly conserved, WD repeat protein, Skb15, which negatively regulates Shk1 in fission yeast. A null mutation in the skb15 gene is lethal and results in deregulation of actin polymerization and localization, microtubule biogenesis, and the cytokinetic machinery, as well as a substantial uncoupling of these processes from the cell cycle. Loss of Skb15 function is suppressed by partial loss of Shk1, demonstrating that negative regulation of Shk1 by Skb15 is required for proper execution of cytoskeletal remodeling and cytokinetic functions. A mouse homolog of Skb15 can substitute for its counterpart in fission yeast, demonstrating that Skb15 protein function has been substantially conserved through evolution.     

Isolation of synthetic lethal mutations in the rsm1-null mutant of fission yeast

To identify mutations in genes that are genetically linked to rsm1, we performed a synthetic lethal genetic screen in the fission yeast, Schizosaccharomyces pombe. Four mutations that showed synthetic lethality in combination with the rsm1null allele were isolated from approximately 320,000 colonies and defined in three complementation groups. One mutant (SLrsm1) exhibited a significant accumulation of poly(A)⁺ RNA in the nucleus under synthetic lethal conditions, while the rest had no mRNA export defects. In addition, some genes (spmex67, rae1, or mlo3) required for mRNA export complemented the growth defects of the identified mutants. These results suggest that the isolated mutants contain mutations in genes that are involved in mRNA export and/or pre-mRNA retention.     

Establishment of a cellular axis in fission yeast

Recent studies in fission yeast Schizosaccharomyces pombe reveal how cells establish a cellular axis that specifies domains as the functional 'ends' and 'middle' of the cell. During interphase, dynamic microtubules position the nucleus at the middle of the cell and orientate microtubule 'plus' ends towards the ends of the cell. At the cell ends, the microtubule plus ends might establish a zone of polarized cell growth and actin assembly by depositing factors such as Tea1p. At the cell middle, the nucleus might specify the position of the actin contractile ring and the future cell division site by positioning cytokinesis factors such as Mid1p.     

A mutant of Arp2p causes partial disassembly of the Arp2/3 complex and loss of cortical actin function in fission yeast

The Arp2/3 complex is an essential component of the yeast actin cytoskeleton that localizes to cortical actin patches. We have isolated and characterized a temperature-sensitive mutant of Schizosaccharomyces pombe arp2 that displays a defect in cortical actin patch distribution. The arp2(+) gene encodes an essential actin-related protein that colocalizes with actin at the cortical actin patch. Sucrose gradient analysis of the Arp2/3 complex in the arp2-1 mutant indicated that the Arp2p and Arc18p subunits are specifically lost from the complex at restrictive temperature. These results are consistent with immunolocalization studies of the mutant that show that Arp2-1p is diffusely localized in the cytoplasm at restrictive temperature. Interestingly, Arp3p remains localized to the cortical actin patch under the same restrictive conditions, leading to the hypothesis that loss of Arp2p from the actin patch affects patch motility but does not severely compromise its architecture. Analysis of the mutant Arp2 protein demonstrated defects in ATP and Arp3p binding, suggesting a possible model for disruption of the complex.     

Multistep and multimode cortical anchoring of tea1p at cell tips in fission yeast

The fission yeast cell-polarity regulator tea1p is targeted to cell tips by association with growing microtubule ends. Tea1p is subsequently anchored at the cell cortex at cell tips via an unknown mechanism that requires both the tea1p carboxy-terminus and the membrane protein mod5p. Here, we show that a tea1p-related protein, tea3p, binds independently to both mod5p and tea1p, and that tea1p and mod5p can also interact directly, independent of tea3p. Despite their related structures, different regions of tea1p and tea3p are required for their respective interactions with an essential central region of mod5p. We demonstrate that tea3p is required for proper cortical localization of tea1p, specifically at nongrowing cell tips, and that tea1p and mod5p are independently required for tea3p localization. Further, we find that tea3p fused to GFP or mCherry is cotransported with tea1p by microtubules to cell tips, but this occurs only in the absence of mod5p. These results suggest that independent protein-protein interactions among tea1p, tea3p and mod5p collectively contribute to tea1p anchoring at cell tips via a multistep and multimode mechanism.     

A wat1 mutant of fission yeast is defective in cell morphology

The organization of the actin cytoskeleton plays an integral role in cell morphogenesis of all eukaryotes. We have isolated a temperature-sensitive mutant in Schizosaccharomyces pombe, wat1-1, in which acting patches are delocalized, resulting in an elliptically shaped cell phenotype. Molecular cloning and DNA sequencing of wat1 ⁺ showed that the gene encodes a 314 residue protein containing WD-40 repeats. Cells lacking wat1 ⁺ are slow growing but viable at 25° C and temperature-sensitive for growth above 33° C. At restrictive temperature, wat1-d strains are phenotypically indistinguishable from wat1-1. When combined with a deletion for the wat1 ⁺ gene, cdc mutants failed to elongate at restrictive temperature and exhibited alterations in actin patch localization. This analysis suggests that wat1 ⁺ is required directly or indirectly for polarized cell growth in S. pombe. Wat1p and a functional, epitope-tagged, version of Wat1p can be overproduced without inducing alterations in cell morphology. Received: 18 September 1996 / Accepted: 22 October 1996     

Direct activation of the fission yeast PAK Shk1 by the novel SH3 domain protein, Skb5

The p21-activated kinase (PAK) homolog Shk1 is essential for cell viability in the fission yeast Schizosaccharomyces pombe. Roles have been established for Shk1 in the regulation of cell morphology, sexual differentiation, and mitosis in S. pombe. In this report, we describe the genetic and molecular characterization of a novel SH3 domain protein, Skb5, identified as a result of a two-hybrid screen for Shk1 interacting proteins. S. pombe cells carrying a deletion of the skb5 gene exhibit no discernible phenotypic defects under normal growth conditions, but when subjected to hypertonic stress, become spheroidal in shape and growth impaired. Both of these defects can be suppressed by overexpression of the Shk1 modulator, Skb1. The growth inhibition that results from overexpression of Shk1 in S. pombe cells is markedly suppressed by a null mutation in the skb5 gene, suggesting that Skb5 contributes positively to the function of Shk1 in vivo. Consistent with this notion, we show that Skb5 stimulates Shk1 catalytic function in S. pombe cells. Furthermore, and perhaps most significantly, we show that bacterially expressed recombinant Skb5 protein directly stimulates the catalytic activity of recombinant Shk1 kinase in vitro. These and additional data described herein demonstrate that Skb5 is a direct activator of Shk1 in fission yeast.     

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.