Dissection of in vitro behavior and microtubule association of centriolar satellites

Centriolar satellites are membrane-less granules that localize around the main microtubule organizing center of animal cells, the centrosome. Centriolar satellites interact with microtubules and molecular motors and move around centrosomes in a microtubule-dependent way. They are scaffolded by pericentriolar material 1 (PCM1) protein, which interacts over 200 proteins including the ones mutated in ciliopathies, primary microcephaly, and schizophrenia. In agreement with their disease links, they have been reported to regulate a diverse array of cellular processes such as primary cilium assembly, Hedgehog signaling, ciliary motility, autophagy and proteostasis. They mediate their functions by acting as cellular machines that store, traffic, modify and assemble their residents. Although centriolar satellite functions and molecular mechanism of action are evident, little is known about how they are assembled and maintained. To address this question, I dissected the biophysical the biophysical properties of PCM1 because it is the only essential protein for centriolar satellite assembly and maintenance. Phase separation has emerged as a mechanism by which a variety of membrane-less organelles assemble. Therefore, my central hypothesis was that PCM1 phase separates to form centriolar satellites. Supporting my hypothesis, centriolar satellites are spherical in shape in cells, dissolve during mitosis and exhibit fusion and fission events. To test my hypothesis, I used condensation assays, removal of solubility tag and treatments of manipulating the external conditions of the solvent. Results from these experiments showed that PCM1 assembles solid spherical condensates and scaffold-like mesh networks in vitro. To gain further insight into the PCM1 domains that contribute to its assembly; localization and microtubule association experiments identified a 923-1200 aa. region in central PCM1 that binds and bundles microtubules in vitro and in vivo. The mutant PCM1 lacking the microtubule binding domain was compromised for microtubule association. The microtubule binding fragment of PCM1 formed condensates upon disruption of microtubule network in cells. In in vitro assays, this fragment formed liquid-like condensates in the absence of microtubules. Taken together, these results identify phase separation as a mechanism that contributes to the assembly of the centriolar satellite scaffold and provide insight into how centriolar satellites interact with microtubules.