Evidence for F-actin-dependent and -independent mechanisms involved in assembly and stability of the medial actomyosin ring in fission yeast.
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Cell division in a number of eukaryotes, including the fission yeast Schizosaccharomyces pombe, is achieved through a medially placed actomyosin-based contractile ring. Although several components of the actomyosin ring have been identified, the mechanisms regulating ring assembly are still not understood. Here, we show by biochemical and mutational studies that the S.pombe actomyosin ring component Cdc4p is a light chain associated with Myo2p, a myosin II heavy chain. Localization of Myo2p to the medial ring depended on Cdc4p function, whereas localization of Cdc4p at the division site was independent of Myo2p. Interestingly, the actin-binding and motor domains of Myo2p are not required for its accumulation at the division site although the motor activity of Myo2p is essential for assembly of a normal actomyosin ring. The initial assembly of Myo2p and Cdc4p at the division site requires a functional F-actin cytoskeleton. Once established, however, F-actin is not required for the maintenance of Cdc4p and Myo2p medial rings, suggesting that the attachment of Cdc4p and Myo2p to the division site involves proteins other than actin itself. (+info)
Control of growth and differentiation by Drosophila RasGAP, a homolog of p120 Ras-GTPase-activating protein.
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Mammalian Ras GTPase-activating protein (GAP), p120 Ras-GAP, has been implicated as both a downregulator and effector of Ras proteins, but its precise role in Ras-mediated signal transduction pathways is unclear. To begin a genetic analysis of the role of p120 Ras-GAP we identified a homolog from the fruit fly Drosophila melanogaster through its ability to complement the sterility of a Schizosaccharomyces pombe (fission yeast) gap1 mutant strain. Like its mammalian homolog, Drosophila RasGAP stimulated the intrinsic GTPase activity of normal mammalian H-Ras but not that of the oncogenic Val12 mutant. RasGAP was tyrosine phosphorylated in embryos and its Src homology 2 (SH2) domains could bind in vitro to a small number of tyrosine-phosphorylated proteins expressed at various developmental stages. Ectopic expression of RasGAP in the wing imaginal disc reduced the size of the adult wing by up to 45% and suppressed ectopic wing vein formation caused by expression of activated forms of Breathless and Heartless, two Drosophila receptor tyrosine kinases of the fibroblast growth factor receptor family. The in vivo effects of RasGAP overexpression required intact SH2 domains, indicating that intracellular localization of RasGAP through SH2-phosphotyrosine interactions is important for its activity. These results show that RasGAP can function as an inhibitor of signaling pathways mediated by Ras and receptor tyrosine kinases in vivo. Genetic interactions, however, suggested a Ras-independent role for RasGAP in the regulation of growth. The system described here should enable genetic screens to be performed to identify regulators and effectors of p120 Ras-GAP. (+info)
A new member of the Sin3 family of corepressors is essential for cell viability and required for retroelement propagation in fission yeast.
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Tf1 is a long terminal repeat (LTR)-containing retrotransposon that propagates within the fission yeast Schizosaccharomyces pombe. LTR-retrotransposons possess significant similarity to retroviruses and therefore serve as retrovirus models. To determine what features of the host cell are important for the proliferation of this class of retroelements, we screened for mutations in host genes that reduced the transposition activity of Tf1. We report here the isolation and characterization of pst1(+), a gene required for Tf1 transposition. The predicted amino acid sequence of Pst1p possessed high sequence homology with the Sin3 family of proteins, known for their interaction with histone deacetylases. However, unlike the SIN3 gene of Saccharomyces cerevisiae, pst1(+) is essential for cell viability. Immunofluorescence microscopy indicated that Pst1p was localized in the nucleus. Consistent with the critical role previously reported for Sin3 proteins in the histone acetylation process, we found that the growth of the strain with the pst1-1 allele was supersensitive to the specific histone deacetylase inhibitor trichostatin A. However, our analysis of strains with the pst1-1 mutation was unable to detect any changes in the acetylation of specific lysines of histones H3 and H4 as measured in bulk chromatin. Interestingly, the pst1-1 mutant strain produced wild-type levels of Tf1-encoded proteins and cDNA, indicating that the defect in transposition occurred after reverse transcription. The results of immunofluorescence microscopy showed that the nuclear localization of the Tf1 capsid protein was disrupted in the strain with the pst1-1 mutation, indicating an important role of pst1(+) in modulating the nuclear import of Tf1 virus-like particles. (+info)
Caffeine can override the S-M checkpoint in fission yeast.
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The replication checkpoint (or 'S-M checkpoint') control prevents progression into mitosis when DNA replication is incomplete. Caffeine has been known for some time to have the capacity to override the S-M checkpoint in animal cells. We show here that caffeine also disrupts the S-M checkpoint in the fission yeast Schizosaccharomyces pombe. By contrast, no comparable effects of caffeine on the S. pombe DNA damage checkpoint were seen. S. pombe cells arrested in early S phase and then exposed to caffeine lost viability rapidly as they attempted to enter mitosis, which was accompanied by tyrosine dephosphorylation of Cdc2. Despite this, the caffeine-induced loss of viability was not blocked in a temperature-sensitive cdc2 mutant incubated at the restrictive temperature, although catastrophic mitosis was prevented under these conditions. This suggests that, in addition to S-M checkpoint control, a caffeine-sensitive function may be important for maintenance of cell viability during S phase arrest. The lethality of a combination of caffeine with the DNA replication inhibitor hydroxyurea was suppressed by overexpression of Cds1 or Chk1, protein kinases previously implicated in S-M checkpoint control and recovery from S phase arrest. In addition, the same combination of drugs was specifically tolerated in cells overexpressing either of two novel S. pombe genes isolated in a cDNA library screen. These findings should allow further molecular investigation of the regulation of S phase arrest, and may provide a useful system with which to identify novel drugs that specifically abrogate the checkpoint control. (+info)
Regulation of the start of DNA replication in Schizosaccharomyces pombe.
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Cells of Schizosaccharomyces pombe were grown in minimal medium with different nitrogen sources under steady-state conditions, with doubling times ranging from 2.5 to 14 hours. Flow cytometry and fluorescence microscopy confirmed earlier findings that at rapid growth rates, the G1 phase was short and cell separation occurred at the end of S phase. For some nitrogen sources, the growth rate was greatly decreased, the G1 phase occupied 30-50% of the cell cycle, and cell separation occurred in early G1. In contrast, other nitrogen sources supported low growth rates without any significant increase in G1 duration. The method described allows manipulation of the length of G1 and the relative cell cycle position of S phase in wild-type cells. Cell mass was measured by flow cytometry as scattered light and as protein-associated fluorescence. The extensions of G1 were not related to cell mass at entry into S phase. Our data do not support the hypothesis that the cells must reach a certain fixed, critical mass before entry into S. We suggest that cell mass at the G1/S transition point is variable and determined by a set of molecular parameters. In the present experiments, these parameters were influenced by the different nitrogen sources in a way that was independent of the actual growth rate. (+info)
Functional expression of the plant alternative oxidase affects growth of the yeast Schizosaccharomyces pombe.
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We have investigated the extent to which functional expression of the plant alternative oxidase (from Sauromatum guttatum) in Schizosaccharomyces pombe affects yeast growth. When cells are cultured on glycerol, the maximum specific growth rate is decreased from 0.13 to 0.11 h-1 while growth yield is lowered by 20% (from 1. 14 x 10(8) to 9.12 x 10(7) cells ml-1). Kinetic studies suggest that the effect on growth is mitochondrial in origin. In isolated mitochondria we found that the alternative oxidase actively competes with the cytochrome pathway for reducing equivalents and contributes up to 24% to the overall respiratory activity. Metabolic control analysis reveals that the alternative oxidase exerts a considerable degree of control (22%) on total electron flux. Furthermore, the negative control exerted by the alternative oxidase on the flux ratio of electrons through the cytochrome and alternative pathways is comparable with the positive control exerted on this flux-ratio by the cytochrome pathway. To our knowledge, this is the first paper to report a phenotypic effect because of plant alternative oxidase expression. We suggest that the effect on growth is the result of high engagement of the non-protonmotive alternative oxidase in yeast respiration that, consequently, lowers the efficiency of energy conservation and hence growth. (+info)
Localization and properties of a silencing element near the mat3-M mating-type cassette of Schizosaccharomyces pombe.
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Transcription is repressed in a segment of Schizosaccharomyces pombe chromosome II that encompasses the mat2-P and mat3-M mating-type cassettes. Chromosomal deletion analysis revealed the presence of a repressor element within 500 bp of mat3-M. This element acted in synergy with the trans-acting factors Swi6, Clr1, Clr2, Clr3, and Clr4 and had several properties characteristic of silencers: it did not display promoter specificity, being able to silence not only the M mating-type genes but also the S. pombe ura4 and ade6 genes placed on the centromere-distal side of the mat3-M cassette; it could repress a gene when placed further than 2.6 kb from the promoter and it acted in both orientations, although with different efficiencies, the natural orientation repressing more stringently than the reverse. Following deletion of this element, two semistable states of expression of the mat3-M region were observed and these two states could interconvert. The deletion did not affect gene expression in the vicinity of the mat2-P cassette, 11 kb away from mat3-M. Conversely, deleting 1.5 kb on the centromere-proximal side of the mat2-P cassette, which was previously shown to partially derepress transcription around mat2-P, had no effect on gene expression near mat3-M. A double deletion removing the mat2-P and mat3-M repressor elements had the same effect as the single deletions on their respective cassettes when assayed in cells of the M mating type. These observations allow us to refine a model proposing that redundant pathways silence the mating type region of S. pombe. (+info)
Disruption of the YRB2 gene retards nuclear protein export, causing a profound mitotic delay, and can be rescued by overexpression of XPO1/CRM1.
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Disruption of the YRB2 gene encoding a nuclear Ran-binding protein homologous to Yrb1p/RanBP1 makes Saccharomyces cerevisiae cold sensitive for colony-formation, but not for growth in liquid medium. Schizosaccharomyces pombe Hba1p, which is homologous to Saccharomyces cerevisiae Yrb2p, rescued the cold sensitivity of Deltayrb2 cells. When released from an alpha factor block, Deltayrb2 cells underwent a prolonged delay at the short spindle stage of mitosis with a normal level of Clb/p34(CDC28) kinase activity, but there was no chromosome loss, this being consistent with the finding that Deltayrb2 was synthetic lethal with neither Deltamad1 nor Deltamad3. The cold sensitive colony-formation of Deltayrb2 cells was rescued by both XPO1/CRM1 and GSP1, but not CDC5, carried on a multicopy vector. XPO1/CRM1 rescued Deltayrb2 even in a single copy. Consistent with such a tight functional interaction, Xpo1p/Crm1p directly bound to Yrb2p, but not Yrb1p, and Deltayrb2 cells were found to have a defect in nuclear export signal (NES)-dependent nuclear protein export. From these results together, the ability of Xpo1/Crm1p to export NES-proteins is suggested to be enhanced by both Yrb2p and Gsp1p, and thereby disruption of YRB2 retards nuclear protein export, resulting in the mitotic delay. (+info)