Dissecting centriolar satellite function using inducible genetic tools

Centriolar satellites are membraneless granules that facilitate protein trafficking to the centrosome and primary cilium, but the principles governing their assembly, dynamics, and functional roles remain poorly defined. Here, we introduce two chemogenetic tools to manipulate satellite organization. First, chemically inducible Kin-14 motor protein-based trafficking system acutely concentrates satellite granules at the centrosome, altering client protein organization. This centrosomal foci created by PCM1 and other clients, behave like a solid compartment and does not dissolve during mitosis. Functionally, forced satellite accumulation disrupts mitotic progression, proper spindle formation, and chromosome congression. In our second system, we are utilizing a chemically inducible centriolar satellite dimerization assay to regulate the multimerization state of PCM1. Ligand-induced dimerization changes satellite organization by enlarging the granules and reducing their numbers. Notably, satellite multimerization prevents their mitotic dissolution and disrupts Hedgehog signaling but does not affect cilium assembly. Finally, we applied a high-throughput image-analysis pipeline to patient derived fibroblasts bearing mutations in ciliopathy related genes and showed that there are no significant differences in satellite number, intensity, or spatial distribution compared to control cells. Together, this thesis establishes various chemical assays for acute perturbation of centriolar satellites, demonstrate that dynamicity and multivalency are critical for proper centriolar satellite function, and indicate that satellite mislocalization and content is not a universal feature of ciliopathies.