| Summary: | <p>An intricate network of kinases and phosphatases safeguards the fidelity and timeliness of chromosome segregation in eukaryotes. As the catalytic subunit of the chromosomal passenger complex (CPC), the Aurora B kinase plays a pivotal role within this network by destabilizing improper kinetochore-microtubule attachments and relaying the attachment status to the spindle assembly checkpoint. Intriguingly, Aurora B is conserved even in kinetoplastids, an evolutionarily divergent group of eukaryotes, who lack a canonical spindle checkpoint, and whose kinetochores are composed of a unique set of structural and regulatory proteins. It remains unknown how chromosome segregation and mitotic exit are controlled in kinetoplastids, and whether the CPC contributes to these processes as in other eukaryotes. A deeper understanding of the mitotic regulatory circuitry in kinetoplastids is bound to provide evolutionary insights into fundamental principles of cell division in eukaryotes and can lead to the discovery of druggable targets to combat infectious diseases caused by kinetoplastid parasites (e.g. sleeping sickness, Chagas disease, and leishmaniasis).</p>
<p>In my DPhil work presented in this thesis, I studied the mechanism of recruitment of the CPC to the kinetochore and the function of the Aurora B kinase in regulating chromosome segregation in <em>Trypanosoma brucei</em>, the causative agent of African sleeping sickness. In the first part of my thesis, I identify two kinetoplastid-specific kinesins, KIN-A and KIN-B, as bona fide CPC proteins in <em>T. brucei</em>. I define an assembly pathway for the trypanosome CPC and demonstrate that the KIN-A subunit controls localization of the CPC to kinetochores and to the central spindle during mitosis. By combining ex vivo cross-linking of native protein complexes with structural and cell biological approaches, I reveal that, unlike other eukaryotes that take advantage of histone modifications for centromere recruitment of the CPC, trypanosomes rely on kinetochore proteins to recruit the CPC. I further demonstrate that the kinesin motor activity of KIN-A drives chromosome alignment in prometaphase and translocation of the CPC to the central spindle upon anaphase onset. The data presented in this section is derived from a recent publication entitled ‘Dynamic localization of the chromosomal passenger complex is controlled by the orphan kinesin KIN-A in the kinetoplastid parasite <em>Trypanosoma brucei'</em> (https://doi.org/10.7554/eLife.93522.1), which is currently under revision at <em>eLife</em>.</p>
<p>In the second part of my thesis, I employ an analogue-sensitive approach to show that inhibition of Aurora B activity causes defects in kinetochore-microtubule attachments and arrests cells in metaphase, a phenotype that has not been reported in traditional model eukaryotes. Aurora B phosphorylates several kinetoplastid kinetochore proteins in vitro, including the recently identified Bub1/BubR1-like protein KKT14 (Ballmer et al. 2024, <em>Open Biology</em>, in press). Depletion of KKT14 overrides the cell cycle arrest caused by Aurora B inhibition, while overexpression of a non-phosphorylatable KKT14 construct delays anaphase entry, indicating that the CPC and KKT14 are entangled in an unconventional circuitry controlling mitotic progression and error-free chromosome segregation in trypanosomes. Moreover, I demonstrate using a nanobody-based system that re-targeting the catalytic module of the CPC to the outer kinetochore is sufficient to promote mitotic exit but causes extensive chromosome mis-segregation in anaphase. Finally, I propose a revised description of the unique architecture of the trypanosome kinetochore based on novel TEM data. A paper arising from the data presented in this section is currently under revision at <em>Journal of Cell Biology</em> under the title: ‘An unconventional regulatory circuitry controls anaphase onset and error-free chromosome segregation in trypanosomes’ (doi: 10.1101/2024.01.20.576407).</p>
<p>The third section of this thesis comprises a brief characterization of the Bub1/BubR1- like protein KKT14, showing that KKT14 interacts with the WD40 domain protein KKT15 and promotes accurate chromosome segregation in anaphase. The data presented here are included in a manuscript entitled ‘Kinetoplastid kinetochore proteins KKT14-KKT15 are divergent Bub1/BubR1-Bub3 proteins’ (https://doi.org/10.1101/2024.01.04.574194), which has recently been accepted for publication in <em>Open Biology</em>.</p>
<p>Finally, I present some preliminary data on the protein phosphatase PP1-1. We recently found that PP1-1 localizes to kinetochores in trypanosomes, making it the second kinetochore-resident phosphatase to have been identified in <em>T. brucei</em> (the other being KKIP7). Unlike KKIP7, PP1-1 is essential for cell growth and error-free chromosome segregation.</p>
<p>In summary, the research presented in this thesis establishes the CPC as a master regulator of mitosis in <em>T. brucei</em> and provides important insights into the largely unexplored regulatory machinery governing cell division in kinetoplastids.</p>
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