Phosphoregulation of divergent cell division in Protozoan parasites

Brusini, Lorenzo (2019) Phosphoregulation of divergent cell division in Protozoan parasites. PhD thesis, University of Nottingham.

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Cell cycle machinery comprises a network of regulatory switches and checkpoints that control the progression of nuclear replication, chromosome segregation and cell division in all complex cells. Decades of research – predominantly in animal or fungal models – has uncovered critical components of this network, in particular demonstrating the essential roles played by specific kinases and phosphatases. Several key players in the network are conserved between humans and yeast, leading to a common assumption that the machinery is similar across all eukaryotes. However, animals and fungi represent only a small subsection of the diversity of eukaryotic cell division. Protozoan parasites represent excellent models to study eukaryotic cell division because: i) they are members of eukaryotic groups that diverged from animals/fungi at or near the origin of eukaryotes, ii) they often divide in ways that are different from canonical models, and iii) due to their importance for human health, they represent some of the best studied cells outside of animal/fungal models. Here, I present work on the phosphoregulation of cell division in two significant groups of parasites – the Apicomplexa, which include the causative agent of malaria, and Kinetoplastida, which cause sleeping sickness and Leishmaniasis.

In Plasmodium, I show that the gene encoding a CDK-related kinase Crk5 is required, but not essential, for blood-stages of P. berghei, and that deletion of the gene creates a defect in cell proliferation. This defect is similar to that for the human malaria parasite P. falciparum but also includes the formation of gametocyte-like cells. Crk5 is also required during replication of mosquito-stages of development. Levels of Crk5 are increased in all replicative stages observed, suggesting that the activity of this kinase might be cycling despite the apparent absence of a regulatory cyclin.

In African trypanosomes, I demonstrate that the KKIP7 protein, which D'Archivio and Wickstead found accumulates at metaphase kinetochores, is a PP1-like phosphatase. Orthologs of KKIP7 form part of a family of phosphatases with unusual N-terminal extension that are also present in a wide range of protozoa. Both the N-terminal extension and phosphatase domain of KKIP7 are required for localisation to the kinetochore. Displacing endogenous KKIP7 with a phosphatase-dead mutant causes a defect in cell cycle progression due to delayed entry into anaphase. These data strongly suggest activation of a spindle assembly checkpoint at the highly divergent kinetochores of trypanosomes that had previously been proposed to be absent. In agreement with this, KKIP7 interacts with proteins present at both the inner and outer kinetochore and also components of the anaphase promoting complex. This association was confirmed using a novel implementation of protein correlation profiling of semi-quantitative mass spectrometry data from immunoprecipitation of multiple kinetochore interacting proteins. This method also shows the presence in trypanosomes of a stable kinetoplastid outer kinetochore complex. Localising 4 proteins for which there was no previous localisation data that showed biochemical affinities with the kinetochore outer kinetochore complex identified 2 new kinetochore components – KKIP10 and KKIP11 – and 2 RNA-binding proteins that associate with the kinetochore during mitosis.

Finally, I present the optimization and implementation of a genome-wide screen for modifier genes of a kkip7 mutant. The genetic tools created allow tunable, inducible expression of mutant transgenes in combination with RNA interference. Using improved means of transfection developed in the lab, I have screened ~240 000 mutants. This is the first example of a genome-wide screen for epistasis in trypanosomes and is likely to applicable to a huge number of experimental questions in future.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Wickstead, Bill
Tewari, Rita
Keywords: Eukaryotic cell division; Phosphoregulation; Modifier genes; Mutant transgenes; Trypanosomes
Subjects: Q Science > QL Zoology > QL360 Invertebrates
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 56884
Depositing User: Brusini, Lorenzo
Date Deposited: 26 Apr 2022 08:56
Last Modified: 26 Apr 2022 08:57

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