Animals, fungi, and amoebas require an actomyosin contractile ring at the division site to perform cytokinesis. The contractile ring initiates and guides the invagination of the plasma membrane as it forms new barriers between the nuclei at the cell equator. Defects in the contractile ring can result in misdirected, delayed, or premature cytokinesis, which leads to abnormal chromosome numbers. Aneuploidies resulting from failed cytokinesis sometimes lead to aggressive forms of cancer. This dissertation was motivated by the goal of better understanding the properties of the contractile ring and how it drives cytokinesis. Actomyosin is initially recruited to the cell equator through the coordination of scaffolding factors, actin-binding proteins, and signaling cascades. Subsequently, the sliding of actin filaments by myosin reshapes the resulting meshwork into a compact ring. Once fully assembled, the contractile ring establishes tension, which leads the plasma membrane inward. The primary motor proteins in the contractile ring of animal cells are class-II nonmuscle myosins, which typically function as bipolar filaments. Filament assembly is activated by phosphorylation and plays a central role in myosin function during cytokinesis. However, many underlying processes that regulate contractile ring function are poorly understood. Current models of cytokinesis have been based on mechanistic insights provided by two decades of work in the fission yeast system Schizosaccharomyces pombe. In fission yeast, the class-II myosin Myo2 provides the major source of motor activity in the contractile ring. Myo2 is two-headed and has a rod-like tail, which is consistent with other class-II myosins. Yet, it was unknown whether Myo2 assembles into filaments, or how phosphorylation affects its activity. To investigate these features, recombinant Myo2 was purified from the baculovirus/Sf9 insect cell expression system. Hydrodynamic measurements were used to examine whether Myo2 forms filaments. These sedimentation velocity data gave no indication that Myo2 self-assembles under the typical physiological salt concentrations, which suggests that Myo2 is unlike any class-II myosin known to date. Myo2 was also treated in vitro with its native kinase Pak1. Phosphorylation of Myo2 molecules had no effect on self-assembly, however it reduced actin-binding in motility assays and increased steady-state ATPase rates by two fold. Our results imply that the function and regulation of fission yeast Myo2 during cytokinesis depends on a specific scaffolding scheme at the plasma membrane, which has not been observed in other eukaryotes. Another interest of this dissertation was how the contractile ring is regulated during cytokinesis. We examined one cytokinesis protein, Cyk3, believed to mediate between the ring and extracellular processes. Genetics and live cell imaging analyses indicated that Cyk3 functions through a catalytically-inactive enzyme domain, which implicated Cyk3's involvement in one of the primary cytokinesis signaling pathways. This dissertation sheds new light on core aspects of how fission yeast undergo cytokinesis, especially with respect to the mechanism of Myo2 activity in the contractile ring. Characterizing the physical and enzymatic properties of an essential myosin in a simple organism should provide insights into cytokinesis in higher organisms.

Heterochromatin assembly in fission yeast (Schizosaccharomyces pombe) requires conserved components that mediate RNA interference (RNAi) directed methylation of histone H3 on lysine 9 (H3K9). Fission yeast heterochromatin is mainly found at centromeres, telomeres, and the mating-type locus. At centromeres, transcripts from repetitive elements are processed to siRNAs and RNAi promotes chromatin modification by recruiting the Clr4 methyltransferase. RNAi is not required to maintain silent chromatin at the mating-type locus. This RNAi-directed form of centromeric heterochromatin provides an ideal system for in vivo screening to allow the identification of compounds that inhibit the activity of proteins involved in RNA silencing, chromatin modification and heterochromatin assembly in fission yeast and may inhibit conserved proteins in other organisms. A dominant selectable marker gene system at fission yeast centromeres that reports loss of heterochromatin integrity by increased resistance to G418 in 96-well plate format liquid cultures was developed. The resulting strain was used to screen a nontargeted chemically diverse compound library in vivo to identify compounds that disrupt the integrity of RNAi-directed heterochromatin. Two compounds, Emi1 and Emi14, were identified and found to cause a significant decrease in the level of H3K9 methylation on the outer repeats at fission yeast centromeres. Growth in the presence of Emi1 or Emi14 also caused a reduction in H3K9 methylation levels at the mating-type locus, suggesting that they do not act through RNAi. Consistent with this, Emi1 and Emi14 did not cause a decrease in centromeric siRNA levels. Analyses therefore suggest that Emi1 and Emi14 do not disrupt RNAi but that they inhibit downstream events in chromatin modification and heterochromatin assembly. Cells lacking RNAi due to loss of Dicer (dcr1Δ) or cells lacking the histone deacetylase (HDAC) Sir2 (sir2Δ) retain significant but lower levels of H3K9 methylation on the centromeric outer repeats. When dcr1Δ or sir2Δ cells were grown in the presence of Emi1 or Emi14 a further reduction in H3K9 methylation levels was observed on the outer repeats. This mimics the effect of combining clr3Δ with dcr1Δ or sir2Δ and suggests that Emi1 and Emi14 may interfere with SHREC function. SHREC is a chromatin remodelling complex that includes the HDAC Clr3 and the chromatin remodeler Mit1 and is known to contribute to heterochromatin integrity. Expression profiling performed on Emi1 and Emi14 treated cells confirmed the previous results. The changes in gene expression following Emi1 and Emi14 treatment were compared to known mutants defective in heterochromatin integrity. The profile of expression changes following Emi14 treatment was found to correlate with alterations in the expression pattern observed in cells with SHREC components deleted. No correlation with mutants lacking other HDACs or RNAi components was detected. Emi1 had a weaker correlation with defective SHREC function and thus may also partially inhibit the SHREC complex. Murine erythroleukemia (MEL) cells harbouring a silenced eGFP reporter transgene were used to assess whether Emi1 and Emi14 also affect silencing in mammalian cells. Emi1 was found to disrupt silencing at the eGFP reporter and this correlated with a decrease in H3K9 methylation. Structurally related analogues of Emi1 and Emi14 were selected and tested in the fission yeast assay. Interpretation of the obtained structure-activity relationships allowed identification of the chemical moieties key to Emi1 and Emi14 activity. Overall, an approach was developed to identify two novel small molecule inhibitors of a well-characterized chromatin modification pathway. The SHREC complex was identified as the putative target of these two compounds and structurally related active analogues were identified for them. Importantly, one of the compounds was also active in mammalian cells, highlighting the usefulness of this approach in identifying compounds that affect higher organisms.

Mto1 recruits γ-tubulin to the sites of cytoplasmic microtubule nucleation in the fission yeast Schizosaccharomyces pombe. The regulation of Mto1 localisation is central to re-modelling of the microtubule cytoskeleton during the cell cycle. This thesis describes how Mto1 is recruited to the cell equator during mitosis, thereby establishing the equatorial microtubule nucleation centre (eMTOC). F-actin is found to be required for Mto1 localisation to the cell equator and Mto1 is shown to co-localise with the cytokinetic actin ring (CAR). Yeast 2-hybrid screening and tandem-affinity purification were used to screen for additional proteins required for Mto1 localisation to the equator. Further candidate screening identified Myp2, a type II myosin present in the CAR, as being required for Mto1 localisation to the cell equator. Myp2 is shown to physically interact with Mto1 and to be required for formation of the post-anaphase microtubule array. The regulation of Mto1 localisation to the cell equator was also studied. Time-lapse microscopy reveals that Mto1 localisation to the equator does not require either the anaphase-promoting complex or the septation initiation network, both of which have been previously shown to be necessary for the recruitment of γ-tubulin to the eMTOC. Maintenance of the equatorial CAR has previously been attributed to the postanaphase array. The position of the CAR in the mto1-427 mutant strain, which fails to nucleate a PAA, is shown to be unaltered from wild-type strain during exponential growth, suggesting that the PAA does not centre the CAR during normal growth.

A yeast two-hybrid assay suggested the possibility of an interaction between Cdc4p, a small EF-hand protein essential for cytokinesis, and Pik1p in S. pombe. This interaction was unexpected, as one function of Cdc4p is that of an essential light chain, bound to the first IQ-motif of type II myosins, whereas Pik1p is a phosphatidylinositol 4-kinase. The objective of this work was to analyze the effects of Pik1p lipid kinase activity on the cell cycle of S. pombe. Another goal of this study was to evaluate the functional significance of the interaction between Cdc4p and Pik1p. This was performed by generating two mutants of pik1: one that abolished lipid kinase activity (pik1-D709A) and one that abolished Pik1p Cdc4p-binding activity (pik1-R838A). Pik1p has a conserved IQ-motif in its C-terminal region. A mutation in this site (R838A), homologous to a residue which was mutated in myosin and abrogated the interaction with Cdc4p, prevented the interaction with Cdc4p in a yeast two-hybrid assay and ELISA. An increase in lipid kinase activity was observed in cell extracts upon ectopic expression of pik1-wt from an episome, which was abolished by a mutation in the lipid kinase domain of Pik1p (D709A), but not by the R838A mutation. However, little to no increase in lipid kinase activity was observed upon ectopic expression of pik1-wt and pik1-R838A in a strain carrying a conditionally lethal allele of cdc4 (cdc4-G107S). This mutation in Cdc4p was shown previously to prevent the interaction with Pik1p in yeast two-hybrid assays. Ectopic expression of pik1-wt suppressed cell proliferation, with disruption of actin cytoskeletal structures and contractile ring formation. These results were not observed with the ectopic expression of the pik1-R838A mutant or when pik1-wt was expressed in the cdc4-G107S strain. Ectopic expression of pik1-R838A resulted in cell shortening, likely through inhibition of growth, and many of the short cells showed an accumulation of the expressed Pik1p protein at the cell tips. Formation of the contractile ring appeared unaffected in cells with ectopic expression of the pik1-D709A mutant, but many of these cells had thick or more than one septum, characteristic of a septation defect. The ectopic expression phenotypes were dosage dependent since lower levels of expression greatly reduced the severity of the ectopic phenotypes. Pik1p lipid kinase activity is essential and, based on ectopic expression studies, is required for septation. There is a physical and functional interaction between Cdc4p and Pik1p which is not essential for cell viability, but suggests a role for Cdc4p in phosphoinositide metabolism.

A yeast two-hybrid assay suggested the possibility of an interaction between Cdc4p, a small EF-hand protein essential for cytokinesis, and Pik1p in S. pombe. This interaction was unexpected, as one function of Cdc4p is that of an essential light chain, bound to the first IQ-motif of type II myosins, whereas Pik1p is a phosphatidylinositol 4-kinase. The objective of this work was to analyze the effects of Pik1p lipid kinase activity on the cell cycle of S. pombe. Another goal of this study was to evaluate the functional significance of the interaction between Cdc4p and Pik1p. This was performed by generating two mutants of pik1: one that abolished lipid kinase activity (pik1-D709A) and one that abolished Pik1p Cdc4p-binding activity (pik1-R838A). Pik1p has a conserved IQ-motif in its C-terminal region. A mutation in this site (R838A), homologous to a residue which was mutated in myosin and abrogated the interaction with Cdc4p, prevented the interaction with Cdc4p in a yeast two-hybrid assay and ELISA. An increase in lipid kinase activity was observed in cell extracts upon ectopic expression of pik1-wt from an episome, which was abolished by a mutation in the lipid kinase domain of Pik1p (D709A), but not by the R838A mutation. However, little to no increase in lipid kinase activity was observed upon ectopic expression of pik1-wt and pik1-R838A in a strain carrying a conditionally lethal allele of cdc4 (cdc4-G107S). This mutation in Cdc4p was shown previously to prevent the interaction with Pik1p in yeast two-hybrid assays. Ectopic expression of pik1-wt suppressed cell proliferation, with disruption of actin cytoskeletal structures and contractile ring formation. These results were not observed with the ectopic expression of the pik1-R838A mutant or when pik1-wt was expressed in the cdc4-G107S strain. Ectopic expression of pik1-R838A resulted in cell shortening, likely through inhibition of growth, and many of the short cells showed an accumulation of the expressed Pik1p protein at the cell tips. Formation of the contractile ring appeared unaffected in cells with ectopic expression of the pik1-D709A mutant, but many of these cells had thick or more than one septum, characteristic of a septation defect. The ectopic expression phenotypes were dosage dependent since lower levels of expression greatly reduced the severity of the ectopic phenotypes. Pik1p lipid kinase activity is essential and, based on ectopic expression studies, is required for septation. There is a physical and functional interaction between Cdc4p and Pik1p which is not essential for cell viability, but suggests a role for Cdc4p in phosphoinositide metabolism.

Critical to a cell's survival is its ability to deal with stress by making an appropriate response. This response often takes place in the cytoplasm, which is everything contained within the cell's plasma membrane that is not the nucleus. The cytoplasm is a dynamic environment and its ability to reorganize is essential to the cell's function. This dissertation presents a novel, previously undiscovered state of cytoplasm organization for the model system Schizosaccharomyces pombe, also known as fission yeast. Typically the fission yeast cytoplasm is a fluid-like environment in which endogenous lipid granules subject to thermal fluctuations, move freely as they explore their local surroundings through diffusion. When the cell is in a nutrient depleted environment it is exposed to the stress of advanced glucose starvation. As a result, we find that the cytoplasm undergoes drastic reorganization reminiscent of a phase transition; it is now a solid-like environment in which there is no visible motion. Lipid granules throughout the cell appear to be completely immobilized and are unable to move through the cytoplasm, despite the application of force through optical tweezers. We term this cytoplasmic state the cell frozen state. The cell frozen state is a physiological state, one that the cell can recover from with the addition of fresh nutrients. It is characterized by an anomalous diffusion exponent of [alpha] = 0.23 ± 0.01, which is a significant reduction from the anomalous diffusion exponent [alpha] = 0.66 ± 0.01 found for exponentially growing cells in which there is visible motion. To account for the cell wide immobilization of lipid granules, we hypothesize the formation of a polymer network all through the cytoplasm, and identify septins 1-3 as the most likely filament formers. In addition, we find there is an increase in the number of vacuoles in the cytoplasm during starvation, and propose a vacuole-septin model to describe the cytoplasm reorganization for the cell frozen state. / text

Dephosphorylation of the Cdc2 kinase by the Cdc25 tyrosine phosphatase is the universally conserved trigger for mitotic entry. Cdc25 is also the point of convergence for checkpoint signaling pathways which monitor the genome for damaged DNA and incomplete replication. In addition, Cdc25 is inhibited by a MAP kinase cascade in the event of osmotic, oxidative and/or heat stress. These pathways inhibit cell cycle progression by phosphorylating Cdc25 resulting in its association with 14-3-3 and nuclear export. Although Cdc25 can be observed leaving the nucleus following inhibitory signals it is controversial whether phosphorylation, 14-3-3 binding or export itself is required for checkpoint proficiency. In fission yeast, Cdc25 is phosphorylated in vitro on 12 serine and threonine residues by the effector kinase of the DNA replication checkpoint, Cds1. Nine of these residues reside in the N-terminal regulatory region, while three are found in the extreme C-terminus of the protein. We show here that phosphorylation the nine N-terminal residues, nor any of the 12 in vitro sites, are required for enforcement of the DNA replication checkpoint. In lieu of Cdc25 phosphorylation the phosphatase is rapidly degraded and mitotic entry prevented by the action of the Mik1 kinase, targeting Cdc2. Thus, multiple mechanisms exist for preventing mitotic entry when S-phase progression is inhibited. The three C-terminal in vitro phosphorylation sites have not previously been examined in fission yeast. However, homology exists between the S. pombe protein and the Cdc25 orthologues in humans, Xenopus and Drosophila in this region. We report here that in S. pombe these sites are required to prevent mitotic entry following replication arrest in the absence of Mik1, and in the maintenance, but not establishment, of arrest following DNA damage. Our previous work showed that Cdc25 nuclear import requires the Sal3 importin-β but at the time we were unable to show a direct interaction between these two proteins. The final chapter of this thesis proves physical interaction by co-immunoprecipitation. Cdc25 mutants lacking all twelve putative Cds1 sites show nuclear localization during mitosis in a sal3- background, effectively reversing the cell cycle regulated pattern of accumulation of the phosphatase. / Thesis (Ph.D, Biology) -- Queen's University, 2011-06-20 12:16:15.71

Continuous sensation and reaction to environmental fluctuations is especially critical to the survival of unicellular organisms. Stress response mechanisms are essential for cells during the vegetative and sexual life cycles and quiescence. The Schizosaccharomyces pombe mitotic activator and stress response serine/threonine kinase Ssp1 acts independent of the major fission yeast Spc1 MAP kinase stress response cascade. Ssp1 is required at high temperatures in the presence of other stressors, ensures long-term viability in quiescent cells and allows efficient cell division in low-glucose conditions. Ssp1 is cytoplasmic but briefly localizes to the cell membrane after exposure to extracellular stress. It plays a role in actin depolymerization and is required for the change of growth polarity after cell division. After identifying 14-3-3 proteins Rad24 and Rad25 as putative Ssp1 binding partners, we confirmed the interaction with co-immunoprecipitation. Association of Ssp1 with Rad24 diminishes after 15 minutes of hyperosmotic stress, however Rad25 binding is retained. Loss of the rad24 gene product rescues both ssp1- mitotic delay at elevated temperatures and sensitivity to 0. 6M KCl. Conversely, overexpression of rad24 exacerbates ssp1 stress sensitivity and mitotic delay. Diffuse actin polarity and spheroid morphology in rad24- cells improves in an ssp1- background. Ssp1 localization to the cell membrane is negatively regulated by Rad24. Ssp1 does not co-localize with Arp3C (actin-related protein 3 homologue C) after osmotic stress, but instead appears to form a ring around the cell, suggesting localization to fission scars. Ssp1 is basally phosphorylated and hyperphosphorylated after glucose deprivation. Ssp1 is shuttled in and out of the nucleus and accumulates in the nucleus in an exportin Cmr1 dependent manner. Ssp1-GFP levels are constant in all stages of the vegetative cell cycle and Ssp1-GFP is present in both the sexual life cycle and quiescence. C-terminal and N-terminal truncation of ssp1 alters its subcellular localization. The C-terminal region is the site of hyperphosphorylation following glucose deprivation and is also necessary for membrane localization following osmotic stress. / Thesis (Ph.D, Biology) -- Queen's University, 2012-01-24 09:49:58.225

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京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18626号 / 生博第317号 / 新制||生||42(附属図書館) / 31526 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 上村 匡, 教授 西田 栄介, 教授 James Hejna / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DGAM