Centrioles are small organelles composed of a 9-fold symmetric array of stabilized microtubules. Centrioles organize a proteinaceous matrix called the pericentriolar material (PCM) to form centrosomes. During the transition from interphase to mitosis, the PCM is remodeled in a process called centrosome maturation that prepares centrosomes to catalyze microtubule generation for spindle assembly.
In Aim 1, we capitalize on our C. elegans expertise to elucidate the pathways that remodel the centrosome during mitotic entry. Surprisingly, our preliminary results suggest that the essential function of Plk1 during centrosome maturation is not its previously documented role in matrix expansion, but the generation of specialized mitotic g-tubulin complex docking sites that enable spindle assembly. We will take a biochemical approach to confirm this finding. In addition, we will determine how the interphase PCM is organized on the outer centriole wall and how it serves as a structural foundation for assembly of the mitotic PCM. To examine the roles of centrioles, my lab collaboratively developed a specific, potent inhibitor of the Plk4 kinase that controls centriole duplication, called centrinone. Work using centrinone to deplete centrioles from cells, has led us to discover a ubiquitin ligase called TRIM37 that limits the assembly of centrosome-based microtubule-nucleating material to the vicinity of the centriole and controls the susceptibility of cancer cells to Plk4 inhibition. Our preliminary work suggests that TRIM37 may perform these functions by ubiquitinating Plk4 to limit its self-assembly.
In Aim 2, we will rigorously test this hypothesis by performing in vitro assays to determine if TRIM37 can directly ubiquitinate Plk4, and by assessing the effects of this modification on its kinase activity and ability to self-assemble. We will also assess the impact of blocking Plk4 self-assembly on centriole duplication and determine whether TRIM37-based modulation of Plk4 self-assembly controls the efficacy of Plk4 kinase inhibition in cancer cells. The canonical model of the role of the centrosome in spindle assembly is that it contributes to microtubule generation by docking g-tubulin containing complexes on the PCM matrix. Surprisingly, in recent work we discovered that PCM-docked g-tubulin complexes function in parallel to a centriole-based pathway that is sufficient for spindle assembly when the PCM is absent.
In Aim 3, we take candidate and screening-based approaches to understand this previously unappreciated pathway that contributes to microtubule generation during spindle assembly. Collectively, we anticipate that the proposed work will lead to a greater understanding of the PCM remodeling during mitotic entry that allows centrosomes to support spindle assembly and of how Plk4 inhibition might be exploited in the context of cancer therapy.
Centrioles are cellular organelles that are central to the mechanics of cell division and the ability of cells to proliferate. The aims of this proposal are to understand: (1) how centrioles contribute to cell division, and (2) how centriole removal using a chemical inhibitor of a key regulator of centriole duplication may be useful in cancer therapy.
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