Protein topology at the Dictyostelium centrosome

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Centrosomes are the main organizers of microtubules and therefore they play a crucial role in the maintenance of cell architecture and cell cycle progression. They root back to the last eukaryotic common ancestor. Hence they are found in most eukaryotic supergroups, although with rather divergent appearances. Organisms harboring cilia or flagellae in at least one cell type possess centrosomes with centrioles, which are also capable of functioning as basal bodies in cilia formation. In contrast, organisms lacking cilia for locomotion such as amoebozoans or many fungi, usually contain centrosomes without centrioles. As a representative of the latter group, we investigate the centrosome (also called nucleus-associated body) in Dictyostelium amoebae, a very useful species to study those centrosomal functions that are independent of centrioles. Rather than having centrioles, the spheroid Dictyostelium centrosome consists of a three-layered core structure, embedded in a matrix containing microtubule-nucleation complexes, the corona. We have characterized most if not all structural centrosomal components on a molecular level. However, while most individual components have been successfully allocated either to the core or the corona structure, the elucidation of subcentrosomal protein topology and mutual protein interactions is still in its infancy. In our work program we will address the following questions: - Which protein(s) mediate the interaction of the outer layers of the core structure with the corona?- Which protein(s) mediate the interaction of the outer layers with the central layer?- Which proteins interact with the -tubulin-containing nodules within the corona, and/or act as scaffolding proteins?To answer these questions, we will combine proximity-dependent biotin identification (BioID) to map protein-protein interactions on the biochemical level, with superresolution light microscopy (STED and dSTORM) and electron microscopy to draw a detailed topological map of protein localizations at the centrosome. In order to circumvent the need for highly specific antibodies for each protein, and to improve the resolution of our microscopic methods, we will create knock-in strains in which the endogenous centrosomal proteins of interest are completely replaced with tagged variants expressed under control of the endogenous promoter. These tags will be visualized by fluorescent tag-specific nanobodies, or, in case of EM, with Ni-NTA nanogold or gold-labeled nanobodies.