Deciphering the Role of Kinetochores and Microtubules During Interphase and Mitosis in Toxoplasma Gondii

Thesis advisor: Marc-Jan GubbelsThe 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