PROJECT SUMMARY/ABSTRACTCentrosomes are organelles used to build microtubule-based protein machines including mitotic spindles andcilia. At the centrosome core lies a pair of `mother-daughter' centrioles barrel-shaped structures that act as theduplicating elements of the organelle. Normally the centriole pair duplicates only once each cell cycle andduring mitotic entry centrioles recruit a shell of pericentriolar material (PCM) a process called `maturation' from which microtubules grow. Not only are they one of the largest protein complexes in eukaryotic cells butone of the most ancient of organelles and have fascinated cell biologists since their discovery in the late 19thcentury. During the past 20 years advances in imaging proteomics and functional genomic screens have ledto an explosion of discoveries in the centrosome field. At present we have a complete inventory of the proteinscomprising centrosomes. In our model system Drosophila centrosomes assemble from a surprisingly smallnumber of components (approximately 20). Despite these advances many important questions remainunanswered. Although only two conserved master-regulators Polo kinase and Polo-like kinase 4 (Plk4) initiatecentriole maturation and duplication respectively it is not known how they are activated specifically oncentrioles. Also what are the phosphorylation targets of these kinases and how do they promote centrioleduplication and maturation? How are mother centrioles restrained to spawn only a single daughter once per cellcycle? How is centriole length controlled? Understanding these processes at the molecular level is importantbecause alterations in centrosome function or number cause a number of serious pathologies including birthdefects ciliopathies and cancer. Plk4 has been the centerpiece of our research program because it is bothnecessary and sufficient to induce centrosome overduplication (amplification) when overexpressed a scenarioobserved in cancer cells. We have published a series of studies that have defined Plk4 regulation and identifiedseveral of its substrates. Notably Plk4 utilizes multiple mechanisms of control to restrain its activity and preventrampant centrosome overduplication using an elaborate combination of autophosphorylation ubiquitination andautoinhibition. We continue to pursue two overarching goals: 1) identifying the molecular mechanisms thatsuppress centrosome amplification (funded by R01 GM110166) and 2) characterizing the inherent mechanismsthat govern centrosome function and duplication (funded by R01 GM126035). Building on our progress duringthe past five years we propose to extend our studies that will define the mechanisms underlying the fivesequential steps in the assembly process. Specifically we will determine (i) how a single site of daughtercentriole assembly is selected on mother centrioles (ii) the composition of the pre-procentrioles and how it forms(iii) how nascent daughter centrioles assemble (iv) how centriole growth is controlled and (v) the initial steps incentrosome maturation.