Thursday, December 26, 2024
12/26/2024
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) – a process called `maturation' – 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 20 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. Although only two conserved master-regulators, Polo kinase and Polo-like kinase 4 (Plk4), initiate centriole maturation and duplication, respectively, it is not known how they are activated specifically on centrioles. Also, what are the phosphorylation targets of these kinases and how do they promote centriole duplication and maturation? How are mother centrioles restrained to spawn only a single daughter once per cell cycle? How is centriole length controlled? Understanding these processes at the molecular level is important because alterations in centrosome function 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 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: 1) identifying the molecular mechanisms that suppress centrosome amplification (funded by R01 GM110166) and 2) characterizing the inherent mechanisms that govern centrosome function and duplication (funded by R01 GM126035). Building on our progress during the past five years, we propose to extend our studies that will define the mechanisms underlying the five sequential steps in the 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 and how it forms, (iii) how nascent daughter centrioles assemble, (iv) how centriole growth is controlled, and (v) the initial steps in centrosome maturation.
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