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Abstract

The centrosome is an organelle responsible for the separation of DNA during mitosis in most animal cells. It consists of a pair of microtubule-based centrioles surrounded by a layer of proteinaceous Peri-Centriolar Material (PCM), responsible for polymerizing and organizing microtubules, especially in the context of the mitotic spindle. Despite decades of research, the fine molecular architecture of the centrosome, and the PCM in particular, has remained unclear. The nematode C. elegans, having relatively simple centrosomes, is a good well-characterized model for their study. A minimal C. elegans centrosome has even been reconstituted in vitro, with microtubules being concentrated from purified centrosomal protein into so-called asters. In this thesis, I aimed to characterize the structures within C. elegans centrosomes, and in doing so better understand this key organelle. To this end I made use of cryo-electron tomography (cryo-ET), which can reveal cellular structures at molecular resolution in their native state.

The data in this thesis shows that microtubule asters, although posing interesting questions about how microtubules form, are insufficient on their own to act as a model for centrosomal function. Simultaneously, I demonstrate that embryonic cells dissociated from the native embryo remain viable and are a suitable sample for cryo-ET analysis, as they are small enough to be vitrified through plunge freezing and can then be milled down to the desired thickness through use of a Focused Ion Beam (FIB). I then make use of correlative light approaches to find the centrosome inside these milled cells. Within the centrosome, I see a high level of microtubule organization, likely templated by the function of gamma-TuRC complexes which were averaged into a coherent structure. The centrioles seen within these centrosomes contained unexpected features and structural variation, which hints at the specification of their biological functions. The centrosomes themselves were divided into architectural zones: with centrioles at the heart of the organelle, surrounded by a microtubule excluded zone and then a ribosome excluded zone. These zones contained a gel-like meshwork of PCM which functions as a selective barrier for centrosomal client proteins. Thus, my thesis builds a three-dimensional structural atlas that helps explain how centrosomes assemble, grow, and achieve function.