The centriole is a major organelle in eukaryotes, responsible for formation of a polarized cytoskeleton, the bipolar mitotic spindle, and cilia, both motile and primary. Defects in centriole dependent processes result in a wide spectrum of developmental diseases caused by chromosome missegregation, cell migration defects, and abnormal cilia formation including, but not limited to, Down syndrome, retinitis pigmentosa, polydactyly, situs inversus, hydrocephalus, obesity, and Bardet-Biedl syndrome. Centriole biogenesis is tightly coupled to the cell cycle, regulated by a host of factors that ensure centriole duplication is licensed once and only once per cell cycle. A master licensing factor responsible for centriole duplication is the polo-like kinase 4 (Plk4). Plk4's mechanism of action and the role a central conserved domain in Plk4 plays to modulate kinase specificity and kinase activity remains to be determined. This application develops the hypothesis that Plk4's unique central conserved domain plays a critical role in modulating kinase activity and substrate recognition. Three series of experiments examine the role of Plk4's central conserved domain in modulating kinase activity and substrate recognition in relation to centriole duplication. Studies will focus on Drosophila Plk4 given the high level of research conducted on Drosophila Plk4 and the power and ease of translating findings into Drosophila developmental studies. The high degree of identity between human and Drosophila Plk4 facilitates the transfer of Drosophila Plk4 mechanistic insight directly to human Plk4 function. Thus, Specific Aim 1 is to define, at atomic resolution, the structure of Plk4's central domain and the determinants responsible for kinase domain binding. X-ray crystallography will be employed to determine the structure of the central domain alone and in complex with the kinase domain.
Specific Aim 2 is to ascertain the role Plk4's central conserved domain plays in centriole duplication. High resolution fluorescent imaging techniques will be employed to examine the localization of Plk4 constructs and the effect these constructs have on centriole duplication. This examination will use Drosophila S2 cells depleted of endogenous Plk4 using RNAi.
Specific Aim 3 is to determine the role of Plk4's central conserved domain on kinase activity. This study will use standard kinase assays to measure the activity of Plk4 constructs that include or lack the conserved central domain. The long term objectives of this investigation are to ascertain the mechanism of Plk4-dependent centriole duplication, identify centriole-specific Plk4 binding and phosphorylation targets, as well as determine the downstream effectors these Plk4 targets interact with. A fundamental understanding of centriole biogenesis, identifying regulatory components and therapeutic targets, will enhance our understanding of centriole biogenesis in human health and development. Ameliorating defects in centriole-dependent processes, including bipolar mitotic spindle formation, cell migration, and ciliogenesis, will have far reaching medical implications in the development of healthy newborn children and the healthy, independent, and productive functioning of adults.
The primary cilium is a cellular organelle, nucleated off a centriole, which extends away from the cell like an antennae to receive extracellular signals ranging from fluid flow to light and small molecules. Ciliopathies, due to defects in the primary cilia, manifest in a range of disorders that effect human health, including randomization of left-right body axis, abnormalities in neural tube closure, polydactyly, obesity, cystic kidney disease and blindness, among others. The proposed research aims to examine the molecular mechanism of centriole biogenesis, which affords the nucleating template for correct and accurate formation of the primary cilia;the research will impact public health by identifying potential targets for medical intervention where normal cilia formation is defective.
| Slevin, Lauren K; Nye, Jonathan; Pinkerton, Derek C et al. (2012) The structure of the plk4 cryptic polo box reveals two tandem polo boxes required for centriole duplication. Structure 20:1905-17 |
