Centriolar satellites were discovered more than 60 years ago as structures that localize and move around centrosomes. In contrast to our extensive understanding of the biology of centrosomes, centriolar satellites have remained as poorly characterized membrane-less organelles. The overall objective of the CentSatRegFunc project was to address the key unknowns about centriolar satellites, which are their components and interactions, their function and their relationship with centrosomes and cilia. As part of Objective 1, we tested the function of satellites as cellular machines for trafficking. To this end, we developed live imaging assays and single particle tracking algorithms and determined the dynamic behavior of centriolar satellites in cells. In contrast to simple trafficking model that has been proposed, we showed that satellites combine both trafficking and storage to regulate the composition of centrosomes and cilia. As part of Objective 2, we combined novel proteomics approaches with purifications of satellites. The proteomics approaches rely on identifying all proteins in 20 nm proximity of the protein of interest. Using these approaches, we identified that spatial interactome for a number of known satellites proteins. This interactome informed hypothesis about the functions of these structures. Moreover, they also identified new components of satellites that were previously linked to genetic developmental diseases. By focusing on several of these proteins, we revealed mechanisms that contribute to retinal degeneration and microcephaly. In particular, we showed that retinal degeneration mutations lead to defects in cilium assembly and ciliary protein targeting and defects in centriole duplication and mitosis contribute to microcephaly. In Objective 3, we used genome editing and organelle mispositioning approaches to generate cells devoid of satellites and to misposition satellites to the cell periphery, respectively. Phenotypic characterization of these cells identified satellites as key regulators of cilium assembly, regulation of cilium content and tissue architecture. Unexpectedly, constitutive loss of satellites was dispensable for centrosome duplication, cell proliferation and cell cycle progression. Together, the results from the CentSatRegFunc project defined centriolar satellites as cellular machines where proteins are assembled into multicomponent complexes, modified, stored and/or trafficked and showed that they play critical roles during cellular processes required for development and differentiation.
