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Centriole and basal bodies are related nine-fold symmetric microtubule-based eukaryotic organelles central to the organisation of cilia/flagella and centrosomes. Mechanisms of eukaryotic centriole and basal body assembly are mainly based on studies in animal systems. To understand which centriolar proteins are the universally important ones in the assembly across eukaryotes, a bioinformatic survey presented here investigates the distribution of centriolar and cilia-associated proteins across a diverse range of eukaryotes. This analysis showed also that the basal body function is ancestral to eukaryotes, whereas centrosomal components are specific to Holozoa (which include animals). It also suggested that the ancestor of all eukaryotes possessed a cilium/cilia not only with motility function but also with a sensory role. The most frequently conserved proteins in extant ciliated eukaryotes found in this analysis included SAS-6, SAS-4 and WDR16. To test whether these proteins are also important for basal body assembly in distantly-related species to metazoan and other model organisms where the proteins have been studied to date, the proteins were investigated in Trypanosoma brucei. I used a combination of genetic tools and microscopy techniques to demonstrate that SAS-6 but not SAS-4 is essential for basal body assembly in T. brucei. I showed that WDR16 is a stably integrated component of the transition zone and axoneme but not the basal body. Furthermore, I identified a novel SAS-6 like protein which localises to a position consistent with the basal plate and has the capacity to form into filaments. This thesis provides new insights into the evolution of centrioles and basal bodies, and into the function of conserved centriole proteins in T. brucei, a distantly-related organism to animals.

Centrioles and basal bodies with their characteristic 9+2 structure are found in all major eukaryotic lineages. The correlation between the occurrence of centrioles and the presence of cilia/flagella, but not centrosome-like structures, suggests that the ciliogenesis function of centrioles is ancestral. Here, it is demonstrated that the centriole domain of centrosomes emerged within the Metazoa from an ancestral state of possessing a centriole with basal body function but no functional association with a centrosome. Centrosome structures involving a centriole are metazoan innovations. When an axoneme is still present but no longer fully functional, such as the sensory cilia of Caenorhabditis elegans or, as depicted here, the flagellum of the intracellular amastigote stage of the Leishmania mexicana parasite, the basal body structure is less constrained and can depart from the canonical structure. A general view has emerged that classifies axonemes into canonical motile 9+2 and noncanonical, sensory 9+0 structures. This study reveals this view to be overly simplistic, and additional axonemal architectures associated with potential sensory structures should be incorporated into prevailing models. Here, a striking similarity between the axoneme structure of Leishmania amastigotes and vertebrate primary cilia is revealed. This striking conservation of ciliary structure, despite the evolutionary distance between Leishmania and mammalian cells, suggests a sensory function for the amastigote flagellum. Adding weight to a sensory hypothesis, close examination of Leishmania positioning inside the parasitophorous vacuole revealed frequent contact between the flagellum tip and the vacuole membrane. A sensory function could also explain the retention of a flagellum in Trypanosoma cruzi amastigotes, an intracellular stage that, as shown in this study, emerged independently to the Leishmania amastigote. Basal body appendages, such as pro-basal bodies and microtubule rootlets, also vary widely in their structure. Choanoflagellates, a sister group to the Metazoa, posses an extensive microtubule rootlet system that provides support for their characteristic collar tentacles. This atypical structure is reflected in the underlying molecular components of the choanoflagellate basal body. The importance of choanoflagellates as the closest known relative of metazoans was first revealed by their similarity to choanocytes, the feeding cells of sponges. Although phylogenetic analyses leave little doubt that choanoflagellates are a sister group of animals, comparisons of molecular and structural components of appendages associated with the collar tentacles highlight significant differences and questions the extent to which the collar structures of choanoflagellates and choanocytes can be assumed to be homologous. Finally, the confinement of a centriole-based centrosome to the Metazoa provides little support for the flagellar synthesis constraint as an explanation for the origin of multicellularity. There is, indeed, an apparent constraint; no flagellated or ciliated metazoan cell ever divides. This constraint, however, did not arise until after the incorporation of centrioles into the centrosome in the metazoan lineage and the co-option of centrioles as a structural and functional component of the centrosome. The flagellar synthesis constraint is therefore not an explanation for the origin of multicellularity but a consequence of it.

Centrosomes are the main microtubule organizing centres in animal cells and are formed by a pair of centrioles together with surrounding pericentriolar material (PCM). Cycling cells duplicate their centrosomes strictly once per cell cycle. This process is driven by the semi-conservative duplication of the centrioles that are found at the centrosome core. During the exit from mitosis the two centrioles within the single inherited centrosome separate, and upon the start of S-phase each of these inherited mother centrioles assembles an adjacent daughter at its side. This process results in two complete centrosomes that can form the poles of the mitotic spindle, and thus segregate evenly to the next cell generation. The formation of a daughter centriole suppresses the initiation of new duplication events from the same templating mother centriole until this daughter separates - disengages - at the end of the cell cycle. This regulation - that acts to repress centriole amplification - is summarized in the 'licensing model of centriole duplication' (Tsou and Stearns, 2006). This model states that centriole disengagement provides the license for the re-duplication of mother centrioles. Importantly, experiments show that while abolishing centriole engagement is sufficient to allow mother centrioles to re-duplicate within the same cycle, it is insufficient to allow daughter centrioles the assembly of a granddaughter before they mature into mothers towards the end of their first cell cycle. The molecular nature of this daughter-to-mother transition remains mysterious. In this thesis I show that in Drosophila embryos the essential centriole duplication protein Asl is not incorporated into daughter centrioles as they assemble during S-phase, but is only incorporated once mother and daughter separate at the end of mitosis. The initial incorporation of Asterless (Asl) is irreversible, and is dependent on centriolar DSas-4. Crucially, Asl incorporation is essential for daughter centrioles to mature into mothers that can support centriole duplication. I propose that Asl acts as a permanent primary license that allows new centrioles to duplicate for the first time. Once acquired, this primary license is not lost but rather further regulation is taken over by the reduplication licensing mechanism, disengagement. This work extends the previously proposed licensing model to also explain how new centrioles are licensed for their first duplication event.

SAS-6 is the structural core of the forming centriole - a cylindrical protein complex, which is an essential component of the centrosome. Oligomerisation of SAS-6 is crucial for successful centriole duplication and is achieved through two dimerisation domains in the SAS-6 protein; a long C-terminal coiled-coil domain and a globular N-terminal dimerisation domain. As core components of the centrosome, centrioles help facilitate various cellular functions. They are involved in the anchoring of flagella and cilia to the membrane and in coordinating the spindle apparatus during chromosome segregation. A deeper insight into the molecular mechanisms at play in the centriole duplication process would have implications on our understanding of fundamental cell division processes and a number of related diseases. Here the involvement of an unstudied loop region in the C. elegans SAS-6 N-terminal domain dimerisation is described. Combining structural biology, biophysical and computational techniques, the molecular interactions of this loop were explored, contributing to the oligomerisation of SAS-6 at the N-terminal dimer interface. Furthermore, the screening and testing of small molecule inhibitors of the SAS-6 N-terminal domain dimerisation is described, targeting a hydrophobic pocket in the domain. Two candidate compounds are presented as a result of the screens and next steps towards structure based compound design are suggested, based on computational analysis. The search for inhibitory compounds includes a set-up of an in-house virtual screening pipeline, as well as in vitro screening efforts and a new crystallographic structure of the H. sapiens SAS-6 N-terminal domain. By investigating the making and breaking of the SAS-6 N-terminal domain dimerisation, light is shed on so far neglected details of this essential protein-protein interaction and advancements towards a SAS-6 oligomerisation inhibitor described, which could ultimately be used for new approaches in cell cycle research and might open up new avenues for medical research by binding a disease relevant target.

Centrosomes are organelles that promote microtubule growth. Normally, a single centrosome duplicates once each cell cycle to guide assembly of a bipolar mitotic spindle, ensuring that each daughter cell inherits an equal complement of the genome and a single centrosome. Centrosomes are composed of a pair of ‘mother-daughter’ centrioles and, during duplication, each mother centriole assembles one daughter at a single site. However, mother centrioles can inappropriately assemble multiple daughters, thereby generating centriole amplification (or overduplication), resulting in multipolar spindle assembly and, consequently, chromosome missegration - a driving force for chromosomal instability/aneuploidy which induces birth defects, miscarriage, and tumorigenesis. We have elucidated how the cell cycle control program regulates the centriole duplication machinery to limit centriole duplication to one event per cell cycle via the cell cycle-dependent regulation of Ana2/STIL and PLK4 degradation. In the case of the centrosome licensing factor Plk4, we found that autophosphorylation promotes its own destruction during interphase, which is then counteracted by the Protein Phosphatase 2A (PP2A) in complex with its Twins (tws) regulatory subunit during mitosis. This promotes stabilization of Plk4 and thus allows for the licensing of the mother centriole, making it competent to duplicate during the proceeding S-phase. While PP2Atws plays a positive role in regulating Plk4 to promote centriole duplication, we found that PP2A complexed with the Well-rounded (wrd) and Widerborst (wdb) regulatory subunits negatively regulates Ana2 by promoting its degradation to limit centriole duplication. PP2Awrd/wdb dephosphorylates numerous serine/threonine residues residing in Ana2, including several CDK phosphorylation consensus motifs. We found that CDK1/cycA and CDK2/cycE phosphorylate these residues to promote Ana2 stabilization from S-phase, the start of centriole duplication, to M-phase, the start of centriole duplication licensing. Interestingly, we found that the tumorigenic SV40 virus protein Small Tumor Antigen (ST) amplifies centrioles by targeting the PP2A complex to stabilize Plk4 as well as Ana2, underscoring the oncogenic importance of these newly discovered centriole duplication pathways. Finally, we shed insight into the mechanism for centriole amplification upon Ana2 stabilization by showing that Ana2 associates with Plk4 to promote Plk4 kinase activity as well as Plk4 stabilization.

No description available.

No description available.

No description available.

Centrosomes are cellular organelles present in most animal cells, and are formed of two main components: the centrioles and the pericentriolar material (PCM). Centrosomes perform a variety of functions: they are the main microtubule organising centre in the cell, and are important localisation hubs for kinases involved in regulating the cell cycle. Hundreds of proteins are thought to localise to centrosomes, but work in the last decade has narrowed down this list to a handful of proteins that are thought to be essential for centrosome structure and function in Drosophila. Asl, Ana2, DSas-4, DSas-6 and Sak have been identified as essential components for centriole duplication, while Cnn and DSpd-2 are thought to be important in establishing the PCM. However, the relative position of these 7 components in the pathway of centrosome assembly in Drosophila embryos remains elusive, as a genetics analysis of this process is hampered by the absence of centrioles in most mutant embryos for these proteins. In this thesis I elucidate the pathway of centrosome assembly in Drosophila by using SAPs (DSas-6/Ana2 particles that form in Drosophila unfertilised eggs upon moderate expression of DSas-6 and Ana2) as proxy models of centrosomes. I show SAPs are very similar to centrosomes in composition and dynamics but unlike centrosomes are able to form even in the absence of some of the essential centriolar components. SAP analysis in the absence of each of the main centrosome components reveals that: Sak is not required for the recruitment of downstream components; DSas-4 is necessary for Ana2 and DSas-6 to interact; Asl is the most upstream component of the PCM recruitment pathway, followed by DSpd-2; it is likely that there is an additional PCM recruitment pathway. I then take advantage of some of these results to examine how centrosome formation is potentiated after egg activation. My work allows me to propose an improved description of the pathway of centrosome formation in Drosophila.