Integrative approach to analyze the proximal region of the Trichonympha agilis centriole

The centrosome, the major microtubule organizing center in animal cells, is composed of two orthogonally arranged centrioles and surrounding pericentriolar material. Centrioles are evolutionary conserved organelles characterized by a nine-fold symmetry of microtubules and are built around a cartwheel in their proximal region. The overall architecture at ~ 40 Å resolution of the exceptionally long proximal region of Trichonympha spp. basal body has been uncovered previously using cryo-electron tomography (cryo-ET) followed by subtomogram averaging. The resulting 3D map revealed not only a central cartwheel hub that can accommodate rings of the evolutionarily conserved SAS-6 proteins, but also novel features, whose identity is unknown. In this thesis, we used T. agilis, which is the only Trichonympha sp. with an assembled genome and transcriptomic data, to gain some more insight into the structure, composition and first steps of cartwheel assembly. With the aim of generating a high-resolution 3D model of the T. agilis proximal region, we used cryo-ET and subtomogram averaging to refine the structure of the cartwheel hub and cartwheel inner densities (CID). We uncovered that the CID is polarized, and discovered inter-cartwheel pillars (ICP) between superimposed cartwheel rings. Moreover, we discovered and analysed a new type of the cartwheel of Teranympha mirabilis with a novel type of cartwheel central densities (CCD). We also analyzed the composition of the proximal region of the T. agilis centriole using a proteomic approach. We optimized a density gradient centrifugation protocol for isolation of the T. agilis rostrum, which harbors ~ 1400 centrioles, and performed protein correlation profiling. In this manner, we have identified 47 candidates of the rostrum proteome, including three new T. agilis SAS-6 homologs, which were further characterized. Furthermore, we addressed the oligomerization properties of human and two T. agilis SAS-6 proteins (HsSAS-6, TaSAS-6_1 and TaSAS-6_2) in vitro. We found that HsSAS-6 requires an interaction partner in order to be stabilized and form rings and stacks. In contrast, TaSAS-6_1 did not oligomerize in our setup, and TaSAS-6_2 is able to oligomerize and form different types of oligomers, rings included. In conclusion, the structure and composition of the proximal region of the T. agilis centriole uncovered here are anticipated to be of a general significance for studies of the biogenesis and structure of centrioles.