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Isolation and Characterisation of Regulators of the Septation Initiation Network Regulators in Fission Yeast
No full textThe septation initiation network (SIN) in Schizosaccharomyces pombe regulates cytokinesis. Cdc7p encodes the first kinase in the core SIN pathway. In this study, I performed a genetic screen to identify novel regulators of the SIN pathway by isolating spontaneous revertants of cdc7-24 that displayed a cold-sensitive phenotype. The screen yielded a cold-sensitive allele of ppa2, the major PP2A catalytic subunit, and a mutant in ypa2, one of the two S. pombe PTPA orthologues. I characterised both the PTPA genes in the fission yeast, namely, ypa1 and ypa2. It was determined that both ypa1 and ypa2 are essential for survival at low temperatures. The double null mutant was found to be inviable at all temperatures, indicating that the two gene products have at least one essential overlapping function. It was also established that Ypa2p is the major form of PTPA in S. pombe. Characterisation of the phenotype of the ppa2 and ypa2 mutants suggest that the PP2A complex is involved in establishment of cell morphogenesis, determining site of division plane, the G2/M transition and separation of the divided cells. Genetic interactions of the ypa2 and ppa2 mutants with the SIN mutants suggest that reduction in PP2A activity rescues SIN mutants. It was also determined that ypa2 and ppa2 play a role in regulating asymmetric localisation of Cdc7p during mitosis and association of Sid2p with the contractile actomyosin ring.UPSI
Identification of genetic interactors of ypa2 in Schizosaccharomyces pombe
Get PDFIn this study, the fission yeast Schizosaccharomyces pombe was used as a model system to study the cellular signaling that underlies cell cycle progression. Phosphorylation plays an important part in the regulation of this process. Kinases catalyze the transfer of a phosphate group to a substrate, which may regulate its activity. Protein phosphatases oppose the activity of protein kinases; the balance of their activities regulates signaling flux in many signaling pathways. The Septation Initiation Network (SIN) is a signaling cascade that regulates cytokinesis in fission yeast. SIN signaling is triggered by a GTPase and is transduced by three protein kinases; phosphorylation plays an important role in controlling SIN signalling. Numerous genetic screens and biochemical analyses have identified phosphatases and kinases that regulate the SIN. The protein phosphatases Calcineurin/Protein Phosphatase 2B (PP2B) and Flp1p promote SIN signaling, while PP2A is a SIN inhibitor. PP2A is conserved and is activated by the chaperone Phosphatase Two A Phosphatase Activator (PTPA). S. pombe has two PTPA-related genes, ypa1 and ypa2; the deletion mutant of the latter rescues SIN mutants which cannot undergo cytokinesis, consistent with PP2A opposing SIN signaling. The phenotype of the deletion mutant of ypa2, indicates that it is involved in the control of cell polarity, cell growth, mitotic commitment and cytokinesis. In this study, we characterized the hypomorphic allele ypa2-S2 which rescues SIN mutants, but is otherwise largely normal. ypa2-s2 was used as bait in a synthetic genetic array screen, to identify genes whose products compensate for reduced function of Ypa2p. A large number of hits were identified, of which approximately ninety percent are novel genetic interactors of ypa2. Validation studies indicated that eighty percent of the interactions seen in the high-throughput screen can be reconstructed. The identified genes regulate several biological processes, including signal transduction and protein phosphorylation. This prompted us to further characterize the roles of Ckb1p and Flp1p, in regulating cytokinesis signaling. Ckb1p is a regulatory subunit for Casein Kinase II (CK II), which was previously implicated in polar growth. The phenotype of the mutant ypa2-S2 ckb1-â indicates that Ckb1p, similar to Ypa2p, contributes to the regulation of mitotic commitment and cytokinesis signaling. Furthermore, ckb1-â showed negative genetic interaction with PP2A and SIN mutants. Flp1p is a protein phosphatase that was reported to regulate mitotic commitment and to promote SIN signaling. The double mutant between ypa2-S2 and flp1-â showed cell separation defects and phenotypic analysis of double mutants between flp1-â and PP2A mutants indicates that these phosphatases cooperate in cell separation. Overall, this work has uncovered novel facets of the regulation of cytokinesis in S. pombe, implicating CK II in controlling SIN signaling, and uncovering cooperative interactions between different phosphatases in controlling cytokinesis.UPSI
Isolation and Analysis of Regulators Coordinating the Nuclear Cycle with Cytokinesis in Schizosaccharomyces Pombe
No full textDuring the division of a mother cell into two daughter cells the genetic material of the mother cell is replicated and segregated to the daughter cells before the cytoplasm divides into two portions in a process called cytokinesis. In order to maintain the genetic stability of the daughter cells it is crucial that the bisection of the cells only occurs once the genetic material has been faithfully partitioned. In the model organism S. pombe the timely coordination of cytokinesis is regulated by a cellular signaling cascade called the Septation Initiation Network (SIN). The SIN is required for the formation and contraction of the contractile actomyosin ring (CAR) that assembles in the cell centre and guides the centripetal deposition of septum material during its contraction. Most of the regulators of the SIN are located to the SPB at some stage of the cell cycle. Interestingly, in anaphase B, when the SIN is supposed to be fully active, positive and negative regulators of the SIN localize to opposite SPBs. This asymmetric configuration of the SIN is lost at the end of cytokinesis, when the SIN is turned off; the positive regulators dissociate from the new SPB and the negative regulators assemble on this SPB. In order to understand this process in more detail we analyzed the localization of Cdc7p, a positive regulator of the SIN. We found that the disappearance of Cdc7p from the new SPB at the end of cytokinesis correlated with the separation of the two halves of the cytoplasm. Moreover, our data suggested that the separation of the two SPBs resulting from the completion of septum formation is important for the resetting of the SIN. Interestingly, we observed that Sid2p, the downstream effector of the SIN, was important for the maintenance of asymmetry in anaphase B and for the timing of the resetting of the SIN by acting in a negative feedback. Taken together, we suggested a model where the downstream effector of the SIN may promote the loading of the negative regulators to the SPBs; in anaphase to the old SPB (due to higher affinity of the negative regulators for this SPB) and upon cell separation to both SPBs, because the old SPB cannot buffer the negative feedback. In a separate study we designed a screen to isolate new regulators of the SIN. Mutagenesis of a strain that over-expressed spg1, the core activator of the SIN, led to the isolation of 28 mutants that are dependent on high levels of Spg1p for viability. The characterization of the nine heat-sensitive mutants among them revealed that they are all new alleles of known SIN genes. Interestingly, two mutants that mapped to the SIN effector sid2, lysed at the time of cytokinesis, when shifted to the restrictive temperature. This observation points to a possible role of the SIN in regulating cell separation. Surprisingly, one of the mutants that mapped to the essential positive SIN regulator sid1 contained a premature STOP codon after 243 nucleotides. The analysis of this mutant revealed that translational read-through level of the STOP codon is sufficient for successful cytokinesis. Similarly, high levels of sid1 expression did not interfere with cytokinesis. This indicated that the regulation of the SIN by Sid1p does not depend on its levels but on a precise control of its action. Since over expression of sid1 led to the accumulation of the protein in the nucleus this may hint that Sid1p recruitment to the SPB is regulated by nuclear proteins.UPSI
Metabolic regulation of cytokinesis in Schizosaccharomyces pombe
No full textAll cells need to be able to sense their environment, and adapt their metabolism, growth and cell division appropriately. In this study, I examined the response of Schizosaccharomyces pombe to a change in the carbon source and how that affects cytokinesis. The focus of my work was the Septation Initiation Network (SIN), which is required for contractile actomyosin ring (CAR) assembly and septum formation. The work presented here stems from analysis of the genetic interaction network of etd1. Arguably, Etd1p is the least well-understood component of the SIN and its function in the SIN remains enigmatic. It is not essential at 36°C and at low temperatures, etd1-â dies with an elongated multinucleated phenotype. An extragenic suppressor of an etd1 mutant mapped to pdc101, the main pyruvate decarboxylase isoform in S. pombe. Since fission yeast grows mainly by fermentation in glucose (GLU) medium, Pdc101p plays an essential role the conversion of pyruvate to ethanol and the regeneration of NAD+ in the cytosol. We found that Pdc101p is essential at high temperatures in GLU medium. However, growth in glycerol (GLY) medium, a non-fermentable source, bypassed the requirement for Pdc101p, and surprisingly, Etd1p. GLY medium also rescued, but did not bypass the requirement for, several other SIN mutants. This unexpected result prompted us to investigate how carbon metabolism interacts with the SIN. Glucose-sensing impacts mainly upon the cAMP/PKA pathway in S.pombe. Protein kinase A (Pka1p) is not essential for growth in GLU or GLY medium, though it is required for adjustment to a change of carbon source. Our data revealed that GLY medium decreased the intracellular cAMP level leading to inactivation of Pka1p; pka1-â cells in GLU medium largely phenocopy GLY grown wild-type cells. We found that GLY medium or deletion of pka1 rescued some of the SIN mutants, to the same extent. Our genetic analysis revealed that neither AMP-activated protein kinase nor the Greatwall-Endosulfine pathway is required for the rescue of spg1 mutant by GLY medium or pka1-â . Analysis of etd1-â and etd1-â pka1-â cells in GLU or GLY medium revealed that a significant proportion of etd1-â cells grown on GLU at 36°C (the permissive temperature) exhibit a cytokinesis defect. As expected, this proportion increased significantly at 29°C in GLU medium, but this was rescued if cells were grown in GLY medium or pka1 was deleted. Etd1p has an important role in the association of the SIN proteins with the SPBs, which is vital in the regulation of SIN signalling. Analysis of localisation of the SIN proteins in etd1-â confirmed that Etd1p is essential for Cdc7p localisation in late anaphase and the ring localisation of Mob1p in GLU medium at 29°C. We also found that a significant proportion of GLU-grown etd1-â cells lacked a GFP-Sid1p signal on the SPB during mitosis at both 36°C and 29°C. These defects were completely or partially rescued by growth in GLY medium or pka1 deletion, suggesting a role for Pka1p in controlling SPB association of SIN proteins. Interestingly, forced recruitment of Sid1p to the SPBs was also able to overcome the cytokinesis defects of etd1-â cells, and to rescue some SIN mutants. Our findings reveal novel aspects of SIN regulation and may challenge the current model of the SIN.UPSI
Regulation of constitutive heterochromatin during fission yeast meiosis
Get PDFThe genetic information of eukaryotes is stored in the form of DNA inside the nucleus of the cell. There, the DNA molecules do not exist as naked nucleic acids but are rather wound around histone proteins, forming a higher-level structure called chromatin. Over the years chromatin was categorized into two distinct classes, euchromatin and heterochromatin, using broadly speaking “key features” such as histone modifications or transcriptional activity. Whereas euchromatin is regarded as loose and active, heterochromatin is classically viewed as an inert and silent chromatin state marked by mono-, di-, and tri-methylation of lysine 9 of histone H3 (H3K9me/me2/me3). H3K9 methylation, the hallmark of heterochromatin, plays crucial roles in chromatin-dependent gene silencing and maintenance of genome stability by repressing repetitive DNA elements or recruiting downstream factors essential for faithful chromosome segregation. Many of the fundamental principles and mechanisms of these processes have been elucidated using the fission yeast Schizosaccharomyces pombe (S. pombe) as a model system. Importantly, most of our knowledge about the heterochromatic landscape in organisms, as well as its regulation, results from studies performed in mitotically growing cells. Indeed, spatial and temporal regulation of H3K9 methylation during sexual differentiation has remained understudied even though it is such an important aspect of the cell cycle where precise orchestration of chromatin-related events is needed to prevent aberrant chromosome segregation resulting otherwise in diseases associated with aneuploidy.
The fact that the H3K9 methylation landscape during sexual differentiation of S. pombe was still uncharted area marked the beginning of my PhD project. I set out to map H3K9me2 and H3K9me3 during fission yeast meiosis and I was curious to see whether these modifications change throughout meiotic progression. If so, I wanted to figure out how these changes would be regulated.
I was successful in mapping the two heterochromatic histone marks genome- wide during meiosis and showed that constitutive heterochromatin, especially centromeres, loses H3K9me2 temporarily and becomes H3K9me3 when cells initiate sexual differentiation. This is of particular importance since cells lacking the ability to tri-methylate H3K9 exhibit severe meiotic (but not mitotic) chromosome segregation defects. Trying to understand how such a switch in methylation preference can be achieved, I discovered that the histone lysine methyltransferase (HKMT) responsible for methylating H3K9 in S. pombe, Clr4, is differentially phosphorylated during mitosis and early meiosis and that its phosphorylation state anticorrelates with H3K9me3 activity. Lastly, I identified the cyclin-dependent kinase (CDK) Cdc2 as an enzyme required for Clr4 phosphorylation whose inhibition is a prerequisite for meiotic commitment, coinciding well with the timing of Clr4 dephosphorylation.
In summary, my PhD work revealed the dynamics of constitutive heterochromatin during sexual differentiation and demonstrated that different methylation states of H3K9 are of physiological relevance. What I find fascinating is the fact that Cdc2, a highly conserved master regulator of the cell cycle, is involved in the regulation of the specificity of a HKMT. In general, the high degree of conservation of the proteins involved in my PhD work suggest broad conservation of the mechanism described here. If faithful segregation of chromosomes during meiosis in humans is controlled similarly, this could have key implications for understanding genetic disorders, such as Down syndrome or certain forms of cancer
Pombe's thirteen - control of fission yeast cell division by the septation initiation network
No full textThe 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.UPSI
Dma1-dependent degradation of SIN proteins during meiosis in Schizosaccharomyces pombe
Get PDFThe 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.UPSI
Mid1p/anillin and the septation initiation network orchestrate contractile ring assembly for cytokinesis
No full textIn both animal cells and fungi, cytokinesis proceeds via a contractile actomyosin ring (CAR). Many CAR components and regulators are evolutionarily conserved. In Schizosaccharomyces pombe, the spatial cue for cytokinesis is provided by Mid1p/Anillin, whereas temporal coordination is ensured by the septation initiation network (SIN). However, neither Mid1p nor the SIN is considered to be essential for CAR assembly per se. Here, using 4D imaging, we reveal an unanticipated, novel role for the SIN in CAR assembly. We demonstrate that CAR assembly involves three, genetically separable steps: establishment of a cortical network of CAR proteins, its lateral condensation, and finally, the formation of a homogeneous CAR. We show that SIN mutants fail to form a homogeneous CAR; we identify hypophosphorylation and recruitment of the conserved PCH-family protein Cdc15p to the CAR as critical steps requiring SIN function. Furthermore, we show that in the absence of Mid1p, CAR assembly proceeds via an actomyosin filament, rather than a cortical network of CAR proteins. This mode of assembly is totally dependent on SIN signaling, thereby demonstrating a direct role for the SIN in CAR formation. Taken together, these data establish that Mid1p and the SIN are the key regulators that orchestrate CAR assembly.UPSI
Characterisation of ypa1 and ypa2, the Schizosaccharomyces pombe orthologues of the peptidyl proyl isomerases that activate PP2A, reveals a role for Ypa2p in the regulation of cytokinesis
No full textThe Schizosaccharomyces pombe septation initiation network regulates cytokinesis. Cdc7p is the first kinase in the core septation initiation network; we have screened genetically for SIN regulators by isolating cold-sensitive suppressors of cdc7-24. Our screen yielded a mutant in SPAC1782.05, one of the two fission yeast orthologues of mammalian phosphotyrosyl phosphatase activator. We have characterised this gene and its orthologue SPAC4F10.04, which we have named ypa2 and ypa1, respectively. We find that Ypa2p is the major form of Protein Phosphatase Type 2A activator in S. pombe. A double ypa1-Δ ypa2-Δ null mutant is inviable, indicating that the two gene products have at least one essential overlapping function. Individually, the ypa1 and ypa2 genes are essential for survival only at low temperatures. The ypa2-Δ mutant divides at a reduced cell size, and displays aberrant cell morphology and cytokinesis. Genetic analysis implicates Ypa2p as an inhibitor of the septation initiation network. We also isolated a cold-sensitive allele of ppa2, the major Protein Phosphatase Type 2A catalytic subunit, implicating this enzyme as a regulator of the septation initiation network.UPSI
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