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Thesis advisor: Marc-Jan Gubbels / The obligate intracellular parasite Toxoplasma gondii exhibits closed mitosis, as chromosome segregation occurs with the confines of the nuclear envelope. Distinct structural changes are absent during mitosis, as the nucleolus is maintained and condensation of chromosomes is largely restricted. Moreover, the centromeres are clustered and remain persistently associated with the centrocone (spindle pole). To elucidate the process of chromosome segregation during mitosis in the parasite, the role of kinetochores and microtubules was examined. Localization studies of the functionally conserved kinetochore proteins TgNuf2 and TgNdc80 revealed that clustered kinetochores colocalize with clustered centromeres at the centrocone throughout the cell cycle. Pharmacological disruption of microtubules resulted in partial loss of clustering, which indicates spindle microtubules are necessary, but not strictly required for this process. Furthermore, the generation of a conditional TgNuf2 knockdown revealed this kinetochore protein is essential for chromosome segregation but dispensable for clustering of centromeres, which remain associated with the centrocone. Moreover, in the absence of TgNuf2 the centrosome behaves normally, but looses its association with the centrocone. Further analysis of this phenotype revealed that the centrocone is devoid of spindle microtubules following depletion of this essential kinetochore protein. Examination of tubulin localization dynamics through parasite development showed that the initiation of spindle microtubules occurs at the basal region of the nucleus prior to centrosome duplication. Furthermore, acetylation of α-tubulin, a posttranslational modification associated with microtubule stability, was confirmed to be specifically associated with stabilization of the spindle microtubules following comigration of the centrocone and centrosome to the apical end of the nucleus. Collectively, these data demonstrate that the persistent association of clustered centromeres with the centrocone is independent of spindle microtubules. These discoveries are contributing unprecedented details to chromosome anchoring and segregation during the cell cycle in this protozoan parasite. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

by Ng Wan-yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1980. / Bibliography: leaves 67-88.

Thesis (M.Ph.)--Chinese University of Hong Kong. / Bibliography: leaves 65-86. / Introduction --- p.1-14 / Materials and Methods --- p.15-30 / Results --- p.31-47 / Discussions --- p.48-60 / Conclusion and Summary --- p.61-63 / Acknowledgements --- p.64 / References --- p.65-86 / Tables --- p.87-98 / Figures --- p.99-111

Mitosis and meiosis are different forms of cell division. Mitosis is a non-reductive form of cell amplification whereby DNA chromosomes are replicated and segregated to form two progeny copies of the progenitor cell. Meiosis is a reductive form of cell division creating progeny containing half the chromosome copies of the progenitor cell. Improper chromosome segregation creates aneuploidy, which is poorly tolerated in cells. In cycling mitotic cells, aneuploidy leads to genome instability and cell death. Following meiosis, aneuploidy is associated with infertility, miscarriages, and birth defects. To segregate chromosome copies properly, pairs are physically organized and segregated to progeny cells by a mitotic spindle, whose functionality is tightly regulated. Kinesins are a family of highly conserved dimeric ATPase proteins which; organize spindle shape and size, facilitate chromosome capture and attachment to the spindle, and generate forces which are required for segregation. I investigated the molecular structure and function of human kinesin 13 family protein, Mitotic Centromere Associated Kinesin, MCAK. MCAK is a microtubule depolymerase whose full molecular structure and mechanism of depolymerization is not fully understood. Using in vitro biochemical assays and in vivo TIRF imaging, I found that altering MCAK molecular structure alters MCAK sub-spindle localization and by inference, alters global microtubule dynamics. This study suggests a potential mode for regulating of MCAK activity/function requiring further testing. Compared to over 30 kinesins in humans, showing a large amount of functional redundancy, yeast only has 6 identified kinesins whose function during meiotic cell division are still relatively unknown. I screened the importance and redundancy of yeast kinesins during meiosis. The results suggest similar roles and redundancies in meiosis to that during mitosis, despite different biochemical and biophysical spindle environments. Together, my investigations broaden the understanding of kinesin regulation and functional redundancy during different types of cell division.

At least three major mitotic processes are regulated by the PP2A-B56 phosphatase complex: the Spindle Assembly Checkpoint (SAC), kinetochore-microtubule attachments and sister chromatid cohesion. We show here that these key functions of PP2A-B56, which require its localization to either the kinetochore or centromere, are split between distinct subsets of B56 isoforms. PP2A-B56γ and PP2A-B56δ localize to the outer kinetochore (via BUBR1), whereas PP2A-B56α and PP2A-B56ε localize to the centromere (via Sgo2). The differential localization observed is due to a difference in affinity for the receptors: PP2A-B56γ has a reduced affinity for Sgo2 compared to PP2A-B56α and, vice versa, PP2A-B56α has a reduced affinity for BUBR1 compared to PP2A-B56γ. Given that the known binding interfaces for both BUBR1 and Sgo2 are highly conserved in all B56 isoform, we generated a series of chimeras between B56α and B56γ to uncover isoform specific interactions. This led to the identifications of two distinct regions within B56α and B56γ that regulate the binding to Sgo2 and BUBR1. Furthermore, site directed mutagenesis has revealed that proper holoenzyme assembly has a role in regulating the localization of B56: it is needed for centromeric accumulation and it interferes with kinetochore accumulation of B56α. We will present a model to explain how this differential localization could be linked to post-translational modifications of PP2AC. Together, these results help to clarify how individual PP2A-B56 isoforms achieve subcellular specificity during mitosis.

One form of protein regulation is accomplished by post-translational modification (PTM). In order to test the importance one type of PTM, methylation, in chromosome segregation, we inhibited protein methylation for brief periods in G2 using the general methylation inhibitor adenosine dialdehyde (AdOx). Inhibiting methylation solely in late G2 leads to mitotic defects. We observed that several methylated histone residues; H3K9me3, H4K20me3 and H4K20me1, are predominantly affected by AdOx in G2. We show both that the kinetochore proteins are not affected and that the mitotic checkpoint is intact. Further, we observed structural defects and chromosome misalignment in mitotic cells. These results indicate that methylation events during late G2 operate to maintain and ensure the structural integrity of pericentromeric heterochromatin prior to mitosis. These results suggest that pericentromeric heterochromatin is required for the proper sensing of kinetochore tension and inactivation of the mitotic checkpoint. / Experimental Oncology

Nucleoporins mediate nucleocytoplasmic trafficking in interphase. In mitosis, upon nuclear envelope breakdown, the role and regulation of Nups remain to be elucidated. An important subcomplex of nucleoporins is the Nup107-160 complex, which, in mitosis, is involved in spindle assembly and nuclear pore re-assembly. Here we show that the level of a key constituent of the Nup107-160 complex- Nup96 is cell cycle regulated. We found that the mechanism involved in regulating Nup96 levels in mitosis is proteolysis by the anaphase-promoting complex (APC). Nup96 interacts with the APC, and its proteolysis can be regulated by Cdc20 and Cdh1. Like the Nup107-160 complex, the APC is localized at kinetochores, centrosomes, and spindles. Disruption of Nup96 levels led to an acceleration of prophase to prometaphase transition and, most importantly, resulted in a delay of G1 progression. Thus, regulation of Nup96 proteolysis in mitosis sets the stage for proper G1 progression. Additionally, we have observed differential regulation of members of the Nup107-160 complex during mitosis and have identified interacting partners of Nup96 at the centrosome which reveal a novel role of nucleoporins in regulating microtubule nucleation.

To ensure a continuous supply of phosphate, living organisms have devised complex mechanisms to regulate the uptake and subsequent utilization of this essential nutrient. An important aspect of phosphate metabolism is the storage of excess phosphate as the polymer, polyphosphate. Despite the importance of this polymer to all living organisms, much needs to be learned about its synthesis, storage, or utilization. Furthermore, little is known about the regulatory mechanisms that determine when polyphosphate synthesis or degradation is appropriate. Our work has shown that polyphosphate is a dynamic molecule whose levels fluctuate during the cell cycle. Polyphosphate levels are high in G1, and subsequently drop as the cell uses free phosphate during cell division. Mitotic induction of phosphate regulatory genes, including the acid phosphatase gene PHO5, replenishes polyphosphate levels late in the mitosis. Furthermore, we have shown that Mcm1 and Fkh1, two cell cycle dependent transcriptional activators, contribute to mitotic activation of PHO5. In addition, we have elucidated the importance of regulating polyphosphate synthesis. Strains lacking the cyclin Pho80 have increased expression of the polyphosphate synthase genes, PHM1-4, and thus have highly elevated polyphosphate levels. Hyperaccumulation of polyphosphate results in severe growth defects on medium containing high levels of sorbitol, presumably through the polyphosphate-dependent overacidification of the vacuole.

To ensure a continuous supply of phosphate, living organisms have devised complex mechanisms to regulate the uptake and subsequent utilization of this essential nutrient. An important aspect of phosphate metabolism is the storage of excess phosphate as the polymer, polyphosphate. Despite the importance of this polymer to all living organisms, much needs to be learned about its synthesis, storage, or utilization. Furthermore, little is known about the regulatory mechanisms that determine when polyphosphate synthesis or degradation is appropriate. Our work has shown that polyphosphate is a dynamic molecule whose levels fluctuate during the cell cycle. Polyphosphate levels are high in G1, and subsequently drop as the cell uses free phosphate during cell division. Mitotic induction of phosphate regulatory genes, including the acid phosphatase gene PHO5, replenishes polyphosphate levels late in the mitosis. Furthermore, we have shown that Mcm1 and Fkh1, two cell cycle dependent transcriptional activators, contribute to mitotic activation of PHO5. In addition, we have elucidated the importance of regulating polyphosphate synthesis. Strains lacking the cyclin Pho80 have increased expression of the polyphosphate synthase genes, PHM1-4, and thus have highly elevated polyphosphate levels. Hyperaccumulation of polyphosphate results in severe growth defects on medium containing high levels of sorbitol, presumably through the polyphosphate-dependent overacidification of the vacuole.