Characterizing the molecular mechanisms of centriole duplication and elongation - PROJECT SUMMARY/ABSTRACT Centrosomes are organelles used to build microtubule-based protein machines, including mitotic spindles and cilia. At the centrosome core lies a pair of ‘mother-daughter’ centrioles, barrel-shaped structures that act as the duplicating elements of the organelle. Normally, the centriole pair duplicates only once each cell cycle and, during mitotic entry, centrioles recruit a shell of pericentriolar material (PCM) from which microtubules grow. Not only are they one of the largest protein complexes in eukaryotic cells but one of the most ancient of organelles, and have fascinated cell biologists since their discovery in the late 19th century. During the past 25 years, advances in imaging, proteomics and functional genomic screens have led to an explosion of discoveries in the centrosome field. At present, we have a complete inventory of the proteins comprising centrosomes. In our model system, Drosophila, centrosomes assemble from a surprisingly small number of components (approximately 20). Despite these advances, many important questions remain unanswered. For example, although Polo-like kinase 4 (Plk4) is recognized as the conserved master-regulator of centriole duplication, it is not known how Plk4’s catalytic activity is regulated specifically on centrioles. How are mother centrioles restrained to spawn only a single daughter once per cell cycle? What are the phosphorylation targets of Plk4 and how do the modified components then assemble a centriole? How is centriole length controlled? Understanding these processes at the molecular level is important because alterations in centrosome structure or number cause a number of serious pathologies, including birth defects, ciliopathies and cancer. Plk4 has been the centerpiece of our research program because it is both necessary and sufficient to induce centrosome overduplication (amplification) when overexpressed, a scenario observed in cancer cells. We have published a series of studies that have defined Plk4 structure, regulation and identified several of its substrates. Notably, Plk4 utilizes multiple mechanisms of control to restrain its activity and prevent rampant centrosome overduplication, using an elaborate combination of autophosphorylation, ubiquitination and autoinhibition. We continue to pursue two overarching goals in this renewal of R35 R35GM136265: 1) identifying the molecular mechanisms that suppress centrosome amplification, and 2) characterizing the inherent mechanisms that govern centrosome function and duplication. Building on our progress during the past four years, we propose to continue our studies that will define the mechanisms underlying four sequential steps in the centriole assembly process. Specifically, we will determine (i) how a single site of daughter centriole assembly is selected on mother centrioles, (ii) the composition of the pre-procentrioles, how it forms and its role in centriole duplication, (iii) how Plk4 induces nascent daughter centriole (procentriole) assembly, and (iv) how centriole length is controlled to promote elongation and maintain a terminal length.
