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In Schizosaccharomyces pombe mitosis is initiated when Cdc25 tyrosine phosphatase dephosphorylates Cdc2 (Cdk1) and in turn Cdc2 kinase phosphorylates mitotic targets. Cdc2 is thought to phosphorylate and further activate Cdc25, forming a positive feedback loop between the two for robust entry into mitosis. Pyp3 tyrosine phosphatase is essential in the absence of Cdc25. Its role is thought to be in directly dephosphorylating Cdc2 under these conditions. Pyp3 also presents a link between cell division and growth. It interacts physically and genetically with the mRNA cap-binding protein eIF4E and is thought to play the same role as mammalian 4E-binding proteins. Pyp3 has a consensus TOS motif potentially enabling nutritional input from the TOR pathway into translation regulation. Since known 4E-BPs are not phosphatases, Pyp3 may act as a 4E-binding protein independently of its phosphatase activity. Evolutionarily conserved Cdc2 phosphorylation sites in Cdc25 were substituted to non-phosphorylatable Ala, or to Glu as a phosphomimic. The T89E phosphomimic mutation creates an activated allele of Cdc25, cdc25-89w. It has a dominant semi-wee phenotype due to accelerated entry into mitosis. Pyp3 was mutagenized to remove the function of the phosphatase active site and also the putative TOS motif. The Pyp3 active site is essential for its role in cell cycle initiation. It is also essential for the genetic interaction with eIF4E, tif1. Removal of a putative Pyp3 TOS motif affects the Pyp3 localization to cytoplasmic foci following co-overexpression of eIF4E. Similar localization occurs in response to heat stress. These results make important contributions to the understanding of mitotic initiation, and link between cell growth and division. / Thesis (Master, Biology) -- Queen's University, 2013-09-12 14:40:40.384

Cell-cycle progression in mammalian cells is coordinated by a series of control points. The D-type cyclins are a family of key cell cycle regulators that are controlled largely by mitogens and their association with and activation of cdk 4 and 6 at the G1 phase of the cell cycle. This study seeks to first analyze cyclins D1, D2, and D3 expression patterns in preimplantation mouse embryos using in vivo studies and then analyze the effects of Dilantin on the cyclin D1 expression pattern in cultured embryos. Antibody staining against cyclin D1, D2, and D3 via indirect immunofluorescence using a Zeiss Confocal Microscope and analysis of individual embryo staining intensities using Zeiss computer software were employed to evaluate expression patterns throughout the first three cell cycles. The data showed that all three D cyclins were present throughout the first three cell cycles. Cyclin D1 had peak average fluorescence intensity at the G2 phase of the second cell cycle with a decrease at the G1 in the third cell cycle. Cyclin D2 had a consistent increase of fluorescence intensity throughout all three cell cycles. Cyclin D3 had peak average fluorescence intensity at the G2 phase of the second cell cycle with an immediate decrease at the Gl phase in the third cell cycle. Cyclin D1 was localized to the nucleus in G1 phases of the cell cycle. In contrast, cyclin D2 was found in the nucleus during G2 phases of the cell cycle rather than in G1. Cyclin D3 was not localized to the nucleus in either cell cycle phase throughout the first three cell cycles. These unique nuclear staining patterns seen by D1, D2, and D3 may reflect a function in the cell cycle. Embryos cultured in the presence of l0gg/ml of Dilantin were found to be slowed in development indicated by the absence of transition from the one-cell to the two-cell stage when compared to the controls. Since the Dilantin cultured embryos never reached G1 of the second cell cycle the increase in fluorescence intensity seen was still considered to be a representation of the G2 phase of the first cell cycle. Cyclin Dl's fluorescence intensity was affected by Dilantin and accompanied with unstained nuclei during the G2 phase of the first cell cycle. The peak average fluorescence intensity occurred during the G1 phase of the second cell cycle for cyclin D1 stained CZB control, while the vehicle control, 0.001N NaOH, remained constant. Both CZB and 0.001N NaOH had similar expression patterns seen previously in the cyclin D1 in vivo data. The information gained from the in vivo and in vitro experiments will help to better understand what causes the problems associated with exposure to Dilantin, and also the effects Dilantin has on the cell cycle. / Department of Biology

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The human pathogen Candida albicans is polymorphic, and its ability to switch growth forms is thought to play an important role in virulence. The primary research aim of this thesis was to understand the role the mitotic exit network plays in C. albicans with particular focus on the Tem1 GTPase protein. This aim was split into three specific goals; to study the role of Tem1 through the construction of a regulatable tem1 mutant, to understand the regulation of Tem1 through localisation and protein interaction studies, and to construct new molecular tools utilising the NAT1 positive selection marker in order to achieve two previous goals. In this thesis we demonstrated that TEM1 is an essential gene in C. albicans, and its essential function is signalled through the Cdc15 protein. Surprisingly, Tem1p depleted cells arrested as hyper-polarised filaments containing one or two nuclei and ultimately lost viability. These filaments formed from budding yeast cells, suggestive of a blockage late in the cell cycle. Ultimately the failure of these filaments to undergo cytokinesis was linked to a defect in septin ring dynamics and the formation of actomyosin ring. To understand the regulation of Tem1 we localised both the Tem1 and Lte1 proteins and found that Tem1 localised to spindle pole bodies in a cell-cycle dependent fashion by recruited at the onset of S phase. In contrast, the Lte1 homolog localised to the daughter cell cortex prior to release into the cytoplasm at the end of the cell cycle. A yeast 2-hybrid analysis of the MEN components demonstrated the potential of Bfa1/Bub2 and Tem1 to form a complex and the ability of Tem1 to homodimerise which may play a role in its self-activation. In order to carry out various aspects of this work we constructed a fully functional set of cassettes, including the constitutively active ENO1 promoter, V5-6xHIS epitope tag and various fluorescent protein genes fused to the NAT1 positive selection marker. When considered together, these results indicate that Tem1 is required for timely mitotic exit and cytokinesis in C. albicans, similar to S. cerevisiae, but the final output of the pathway must have diverged.