PLK4 SELF-PHOSPHORYLATION DRIVES THE SELECTION OF A SINGLE SITE FOR PROCENTRIOLE ASSEMBLY
Johns Hopkins University
Centrioles are cylindrical-shaped organelles that recruit a surrounding pericentriolar material (PCM) to form the centrosome, a microtubule-organizing center that arranges the interphase microtubule cytoskeleton and forms the poles of the mitotic spindle during cell division. Each dividing cell inherits a single centrosome from a previous division, but two are required to build the bipolar mitotic spindle successfully. As a single pair of centrioles exists as the core of the centrosome, centriole copy number is directly regulated to ensure faithful cell division. Therefore, each centriole within the pair must duplicate only once every cell cycle. Centriole duplication occurs in S-phase and is regulated by Polo-like kinase 4 (PLK4). How PLK4 selects a single site on parent centrioles for procentriole assembly remains unclear. As centrioles are near the resolution limit of traditional widefield microscopes, studying changes in PLK4 localization has been challenging. To circumvent this, we utilized ultrastructure expansion microscopy (U-ExM) to physically expand centrioles ~4x isotropically and show that PLK4 localizes at discrete sites along the wall of parent centrioles. While there is variation in the number of sites PLK4 occupies, most PLK4 localizes at a dominant site on parent centrioles that directs procentriole assembly. Inhibition of PLK4 stabilizes its binding at the centriole and increases occupancy to a maximum of nine sites, which mirrors the inherent nine-fold rotational symmetry of the centriole. PLK4 kinase activity promotes the release of active PLK4 from the centriole and drives drive the selection of a single site for procentriole assembly. PLK4 self- phosphorylates ten sites along a flexible linker to promote release. Preventing linker phosphorylation through mutation blocks PLK4 turnover, leading to supernumerary sites of PLK4 localization and centriole amplification. Therefore, self-phosphorylation is a major driver of the spatial patterning of PLK4 at the centriole and plays a critical role in selecting a single centriole duplication site.