A role for the carbon source of the cell and protein kinase A in regulating the S. pombe septation initiation network.

The septation initiation network (SIN) is a conserved signal transduction network, which is important for cytokinesis in Schizosaccharomyces pombe. The SIN component Etd1p is required for association of some SIN proteins with the spindle pole body (SPB) during anaphase and for contractile ring formation. We show that tethering of Cdc7p or Sid1p to the SIN scaffold Cdc11p at the SPB, rescues etd1-Δ. Analysis of a suppressor of the mutant etd1-M9 revealed that SIN signalling is influenced by the carbon source of the cell. Growth on a non-fermentable carbon source glycerol reduces the requirement for SIN signalling but does not bypass it. The decreased need for SIN signalling is mediated largely by reduction of protein kinase A activity, and it is phenocopied by deletion of pka1 on glucose medium. We conclude that protein kinase A is an important regulator of the SIN, and that SIN signalling is regulated by the carbon source of the cell.

Pombe's thirteen - control of fission yeast cell division by the septation initiation network.

The septation initiation network (SIN) regulates aspects of cell growth and division in Schizosaccharomyces pombe and is essential for cytokinesis. Insufficient signalling results in improper assembly of the contractile ring and failure of cytokinesis, generating multinucleated cells, whereas too much SIN signalling uncouples cytokinesis from the rest of the cell cycle. SIN signalling is therefore tightly controlled to coordinate cytokinesis with chromosome segregation. Signalling originates from the cytoplasmic face of the spindle pole body (SPB), and asymmetric localisation of some SIN proteins to one of the two SPBs during mitosis is important for regulation of the SIN. Recent studies have identified in vivo substrates of the SIN, which include components involved in mitotic control, those of the contractile ring and elements of the signalling pathway regulating polarised growth. The SIN is also required for spore formation following meiosis. This has provided insights into how the SIN performs its diverse functions in the cell cycle and shed new light on its regulation.

Analysis of S. pombe SIN protein association to the SPB reveals two genetically separable states of the SIN.

The Schizosaccharomyces pombe septation initiation network (SIN) regulates cytokinesis, and asymmetric association of SIN proteins with the mitotic spindle pole bodies (SPBs) is important for its regulation. Here, we have used semi-automated image analysis to study SIN proteins in large numbers of wild-type and mutant cells. Our principal conclusions are: first, that the association of Cdc7p with the SPBs in early mitosis is frequently asymmetric, with a bias in favour of the new SPB; second, that the early association of Cdc7p-GFP to the SPB depends on Plo1p but not Spg1p, and is unaffected by mutations that influence its asymmetry in anaphase; third, that Cdc7p asymmetry in anaphase B is delayed by Pom1p and by activation of the spindle assembly checkpoint, and is promoted by Rad24p; and fourth, that the length of the spindle, expressed as a fraction of the length of the cell, at which Cdc7p becomes asymmetric is similar in cells dividing at different sizes. These data reveal that multiple regulatory mechanisms control the SIN in mitosis and lead us to propose a two-state model to describe the SIN.

Dma1-dependent degradation of SIN proteins during meiosis in Schizosaccharomyces pombe.

The Schizosaccharomyces pombe septation initiation network (SIN) is required for cytokinesis during vegetative growth and for spore formation during meiosis. Regulation of the SIN during mitosis has been studied extensively, but less is known about its meiotic regulation. Here, we show that several aspects of SIN regulation differ between mitosis and meiosis. First, the presence of GTP-bound Spg1p is not the main determinant of the timing of Cdc7p and Sid1p association with the spindle pole body (SPB) during meiosis. Second, the localisation dependencies of SIN proteins differ from those in mitotic cells, suggesting a modified functional organisation of the SIN during meiosis. Third, there is stage-specific degradation of SIN components in meiosis; Byr4p is degraded after meiosis I, whereas the degradation of Cdc7p, Cdc11p and Sid4p occurs after the second meiotic division and depends upon the ubiquitin ligase Dma1p. Finally, Dma1p-dependent degradation is not restricted to the SIN, as we show that Dma1p is needed for the degradation of Mcp6p (also known as Hrs1p) during meiosis I. Taken together, these data suggest that stage-specific targeted proteolysis plays an important role in regulating meiotic progression.

The role of Schizosaccharomyces pombe dma1 in spore formation during meiosis.

Meiosis is a specialised form of the cell cycle that gives rise to haploid gametes. In Schizosaccharomyces pombe, the products of meiosis are four spores, which are formed by encapsulation of the four meiosis II nuclei within the cytoplasm of the zygote produced by fusion of the mating cells. The S. pombe spindle pole body is remodelled during meiosis II and membrane vesicles are then recruited there to form the forespore membrane, which encapsulates the haploid nucleus to form a prespore. Spore wall material is then deposited, giving rise to the mature spore. The septation initiation network is required to coordinate cytokinesis and mitosis in the vegetative cycle and for spore formation in the meiotic cycle. We have investigated the role of the SIN regulator dma1p in meiosis; we find that although both meiotic divisions occur in the absence of dma1p, asci frequently contain fewer than four spores, which are larger than in wild-type meiosis. Our data indicate that dma1p acts in parallel to the leading-edge proteins and septins to assure proper formation for the forespore membrane. Dma1p also contributes to the temporal regulation of the abundance of the meiosis-specific SIN component mug27p.

Functional differentiation of tbf1 orthologues in fission and budding yeasts.

In Saccharomyces cerevisiae, TBF1, an essential gene, influences telomere function but also has other roles in the global regulation of transcription. We have identified a new member of the tbf1 gene family in the mammalian pathogen Pneumocystis carinii. We demonstrate by transspecies complementation that its ectopic expression can provide the essential functions of Schizosaccharomyces pombe tbf1 but that there is no rescue between fission and budding yeast orthologues. Our findings indicate that an essential function of this family of proteins has diverged in the budding and fission yeasts and suggest that effects on telomere length or structure are not the primary cause of inviability in S. pombe tbf1 null strains.

Noninvasive characterization of the fission yeast cell cycle by monitoring dry mass with digital holographic microscopy.

Digital holography microscopy (DHM) is an optical technique which provides phase images yielding quantitative information about cell structure and cellular dynamics. Furthermore, the quantitative phase images allow the derivation of other parameters, including dry mass production, density, and spatial distribution. We have applied DHM to study the dry mass production rate and the dry mass surface density in wild-type and mutant fission yeast cells. Our study demonstrates the applicability of DHM as a tool for label-free quantitative analysis of the cell cycle and opens the possibility for its use in high-throughput screening.

Analysis of the S. pombe Meiotic Proteome Reveals a Switch from Anabolic to Catabolic Processes and Extensive Post-transcriptional Regulation.

Meiotic progression in S. pombe is regulated by stage-specific gene expression and translation, changes in RNA stability, expression of anti-sense transcripts, and targeted proteolysis of regulatory proteins. We have used SILAC labeling to examine the relative levels of proteins in diploid S. pombe cells during meiosis. Among the 3,268 proteins quantified at all time points, the levels of 880 proteins changed at least 2-fold; the majority of proteins showed stepwise increases or decreases during the meiotic divisions, while some changed transiently. Overall, we observed reductions in proteins involved in anabolism and increases in proteins involved in catabolism. We also observed increases in the levels of proteins of the ESCRT-III complex and revealed a role for ESCRT-III components in chromosome segregation and spore formation. Correlation with studies of meiotic gene expression and ribosome occupancy reveals that many of the changes in steady-state protein levels are post-transcriptional.

Cell division: SIN, cytokinesis and ethanol dependency.

A novel mutant screen in fission yeast has identified the 'ethanol dependent' protein etd1p as a potential link between the septation initiation network (SIN), which initiates cytokinesis, and the actomyosin contractile ring that drives separation of the two daughter cells at the end of mitosis.

Plants, MEN and SIN.

In fission yeast, the onset of septation is signalled through the septum initiation network (SIN) signaling pathway. Similarly, in budding yeast the onset of budding is signalled through the mitotic exit network (MEN) pathway. We previously characterized in Arabidopsis signaling elements (GTPases, kinases) closely related to the core elements (spg1p/TEM1p, cdc7p/CDC15p) of the SIN and MEN pathways. Our first results suggested that a plant signaling pathway must be used to coordinate mitotic exit with cytokinesis. This review questioned the value of such an hypothesis in a multicellular organism. The core elements (G-protein, kinase) of the SIN and MEN pathways were only detected in fungi, plants and Mycetozoa. We also noticed that AtSGP GTPase and AtMAP3Kepsilon kinase revealed two paralogues in Arabidopsis. Although Arabidopsis genes complement fission yeast mutants, and Arabidopsis proteins interact with fission yeast proteins, plants do not use these core elements to coordinate the termination of cell division with cytokinesis. Transcriptional regulation and expression data suggest a function for the plant SIN-like elements in the control of cell type specification. Exploring the evolutionary conservation of an ancient signaling pathway provides evidence that evolution has recycled regulatory elements for elaborating a new signaling avenue.

Functional characterization of Pneumocystis carinii brl1 by transspecies complementation analysis.

Pneumocystis jirovecii is a fungus which causes severe opportunistic infections in immunocompromised humans. The brl1 gene of P. carinii infecting rats was identified and characterized by using bioinformatics in conjunction with functional complementation in Saccharomyces cerevisiae and Schizosaccharomyces pombe. The ectopic expression of this gene rescues null alleles of essential nuclear membrane proteins of the Brr6/Brl1 family in both yeasts.

Mitotic hyperphosphorylation of the fission yeast SIN scaffold protein cdc11p is regulated by the protein kinase cdc7p.

The fission yeast septation initiation network (SIN) triggers the onset of septum formation and cytokinesis. SIN proteins signal from the spindle pole body (SPB), to which they bind in a cell cycle-dependent manner, via the scaffold proteins sid4p and cdc11p. cdc11p becomes hyperphosphorylated during anaphase, when the SIN is active. We have investigated the phosphorylation state of cdc11p during mitosis in various mutant backgrounds. We show that association of cdc11p with the spindle pole body is required for its phosphorylation and that ectopic activation of the SIN results in hyperphosphorylation of cdc11p. We demonstrate that mitotic hyperphosphorylation of cdc11p requires the activity of cdc7p and that its dephosphorylation at the end of mitosis requires PP2A-par1p. Furthermore, spindle checkpoint arrest prevents cdc11p hyperphosphorylation. Finally, we show that the septation inhibitor byr4p interacts preferentially with hypophosphorylated cdc11p. We conclude that cdc11p hyperphosphorylation correlates with activation of the SIN and that this may be mediated primarily by cdc7p in vivo.

SIN and the art of splitting the fission yeast cell.

The septation initiation network (SIN) triggers the onset of cytokinesis in the fission yeast Schizosaccharomyces pombe by promoting contraction of the medially placed F-actin ring. SIN signaling is regulated by the polo-like kinase plo1p and by cdc2p, the initiator of mitosis, and its activation is co-ordinated with other events in mitosis to ensure that cytokinesis does not begin until chromosomes have been separated. Though the SIN controls the contractile ring, the signal originates from the poles of the mitotic spindle. Recent studies suggest that the spindle pole body may act as a dynamic assembly site for active SIN signaling complexes. In the budding yeast Saccharomyces cerevisiae the counterpart of the SIN, called the MEN, mediates both mitotic exit and cytokinesis, in part through regulating activation of the phosphoprotein phosphatase Cdc14p. Flp1p, the S. pombe ortholog of Cdc14p, is not essential for mitotic exit, but may contribute to an orderly mitosis-G1 transition by regulating the destruction of the mitotic inducer cdc25p.

Cell Cycle Synchronization of Schizosaccharomyces pombe by Centrifugal Elutriation of Small Cells.

Division of Schizosaccharomyces pombe by medial fission produces identically sized daughter cells that grow by tip extension until their own division is prompted by reaching the same critical size for division as the parental cell. The fidelity of this size control in the absence of perturbation means that cells of the same size are at the same point in the cell cycle. Size selection of small cells from an asynchronous culture by centrifugal elutriation permits generation of synchronous cultures large enough for biochemical analysis. The changes observed in the synchronized cell cycle progression of such cultures are representative of those that accompany cell cycle progression of individual cells. Here, we describe how size selection with the Beckman Coulter JE-5.0 rotor can be used to generate synchronized cultures. Because of the continuous passage of medium through the rotor throughout the procedure, elutriation is considered to have less impact on the integrity of the cell cycle than other approaches. Two protocols are presented here: The first generates a 2-L culture ideal for detailed biochemical analysis, whereas the second allows rapid generation and simultaneous analysis of three smaller (200-mL) cultures.

Cell Cycle Synchronization of Schizosaccharomyces pombe by Lactose Gradient Centrifugation to Isolate Small Cells.

Size selection of small cells from an asynchronous Schizosaccharomyces pombe culture offers a simple way to generate cultures in which progression through the mitotic cell division cycle is synchronized throughout the population. Here, we describe how density centrifugation of cells from asynchronous cultures through lactose gradients selects small G2 cells to generate synchronized cultures as large as 500 mL. The ease and simplicity of this approach makes it an accessible and attractive method for generating synchronous cultures.

Analysis of the Schizosaccharomyces pombe Cell Cycle.

Schizosaccharomyces pombe cells are rod shaped, and they grow by tip elongation. Growth ceases during mitosis and cell division; therefore, the length of a septated cell is a direct measure of the timing of mitotic commitment, and the length of a wild-type cell is an indicator of its position in the cell cycle. A large number of documented stage-specific changes can be used as landmarks to characterize cell cycle progression under specific experimental conditions. Conditional mutations can permanently or transiently block the cell cycle at almost any stage. Large, synchronously dividing cell populations, essential for the biochemical analysis of cell cycle events, can be generated by induction synchrony (arrest-release of a cell cycle mutant) or selection synchrony (centrifugal elutriation or lactose-gradient centrifugation). Schizosaccharomyces pombe cell cycle studies routinely combine particular markers, mutants, and synchronization procedures to manipulate the cycle. We describe these techniques and list key landmarks in the fission yeast mitotic cell division cycle.

Synchronizing Progression of Schizosaccharomyces pombe Cells from Prophase through Mitosis and into S Phase with nda3-KM311 Arrest Release.

Here, we describe how the rapid reversibility of the nda3-KM311 cold-sensitive ß-tubulin mutation was optimized by Mitsuhiro Yanagida's laboratory to synchronize mitotic progression in an entire cell population. The inability to form microtubules following the loss of ß-tubulin function at 20°C triggers the spindle assembly checkpoint, which arrests mitotic progression. Restoration of ß-tubulin function by rewarming to 30°C (or higher) releases the arrest, generating a highly synchronous progression through mitosis. The viability of nda3-KM311 strains at 30°C makes it feasible to generate double mutants between nda3-KM311 and any temperature-sensitive mutant that can also grow at 30°C. These double mutants can be used in reciprocal shift analyses, in which cold-induced early mitotic arrest is relieved by a shift to 36°C, which then inactivates the product of the second mutant gene. The addition of microtubule depolymerizing drugs before the return to 36°C will maintain checkpoint signaling at 36°C transiently, permitting analysis of the impact of temperature-sensitive mutations on checkpoint function. Silencing the checkpoint of nda3-KM311-arrested cells at 20°C through chemical inhibition of aurora kinase is a powerful way to study checkpoint recovery pathways and mitotic exit without anaphase.

Synchronizing Progression of Schizosaccharomyces pombe Cells from G2 through Repeated Rounds of Mitosis and S Phase with cdc25-22 Arrest Release.

Transient inactivation of the cdc25(+) gene product by manipulation of the culture temperature for cdc25-22 cells is the most commonly exploited approach to mitotic synchronization in fission yeast. Because Cdc25 removes the inhibitory phosphate placed on Cdk1 by Wee1, inactivation of Cdc25 arrests cells at the G2/M boundary. Incubation at the restrictive temperature of 36°C for just over one generation time forces all cells in the culture to accumulate at the G2/M boundary. Restoration of Cdc25 function via a return to the permissive temperature or chemical inhibition of Wee1 activity at 36°C can then promote a highly synchronous wave of cell division throughout the culture. These approaches can be performed on any scale and thus support simultaneous assessment of numerous events within a single culture. After describing this simple and widely applicable procedure, we discuss frequently overlooked issues that can have a considerable impact on the interpretation of data from cdc25-22 induction-synchronized cultures.

Peroxiredoxin 1 Protects Telomeres from Oxidative Damage and Preserves Telomeric DNA for Extension by Telomerase.

Oxidative damage of telomeres can promote cancer, cardiac failure, and muscular dystrophy. Specific mechanisms protecting telomeres from oxidative damage have not been described. We analyzed telomeric chromatin composition during the cell cycle and show that the antioxidant enzyme peroxiredoxin 1 (PRDX1) is enriched at telomeres during S phase. Deletion of the PRDX1 gene leads to damage of telomeric DNA upon oxidative stress, revealing a protective function of PRDX1 against oxidative damage at telomeres. We also show that the oxidized nucleotide 8-oxo-2'deoxyguanosine-5'-triphosphate (8oxodGTP) causes premature chain termination when incorporated by telomerase and that some DNA substrates terminating in 8oxoG prevent extension by telomerase. Thus, PRDX1 safeguards telomeres from oxygen radicals to counteract telomere damage and preserve telomeric DNA for elongation by telomerase.

Analysis of the contribution of changes in mRNA stability to the changes in steady-state levels of cyclin mRNA in the mammalian cell cycle.

Cyclins are the essential regulatory subunits of cyclin-dependent protein kinases. They accumulate and disappear periodically at specific phases of the cell cycle. Here we investigated whether variations in cyclin mRNA levels in exponentially growing cells can be attributed to changes in mRNA stability. Mouse EL4 lymphoma cells and 3T3 fibroblasts were synchronized by elutriation or cell sorting. Steady-state levels and degradation of cyclin mRNAs and some other cell cycle related mRNAs were measured at early G1, late G1, S and G2/M phases. In both cell lines mRNAs of cyclins C, D1 and D3 remained unchanged throughout the cell cycle. In contrast, cyclin A2 and B1 mRNAs accumulated 3.1- and 5.7-fold between early G1 and G2/M phase, whereas cyclin E1 mRNA decreased 1.7-fold. Mouse cyclin A2 and B1 genes, by alternative polyadenylation, gave rise to more than one transcript. In both cases, the longer transcripts were the minor species but accumulated more strongly in G2/M phase. All mRNAs were rather stable with half-lives of 1.5-2 h for cyclin E1 mRNA and 3-4 h for the others. Changes in mRNA stability accounted for the accumulation in G2/M phase of the short cyclin A2 and B1 mRNAs, but contributed only partially to changes in levels of the other mRNAs.