Centrioles perform two critical functions in eukaryotes: (1) they direct the accumulation of microtubule nucleating/anchoring material to form centrosomes that play a critical role during cell division, and (2) they serve as basal bodies that direct the formation of microtubule-based cellular projections called cilia, which perform a variety of motile and sensory functions. Centrioles duplicate precisely once per cell cycle in a process that remains poorly understood at a molecular level.
Specific Aims 1 and 2 of the proposed research use the C. elegans embryo as a model system to perform a molecular characterization of the steps in the centriole duplication cycle. Comprehensive RNAi-based screens and genetic analysis in C. elegans have identified 5 proteins, in addition to alpha/beta-tubulin, that localize to centrioles and are required for their assembly. Of these, only two (SAS-4 and SAS-6) have the properties of stably-associated structural components. In the first aim, steps in the duplication cycle will be defined by using fluorescence-microscopy based methods to monitor the kinetics of incorporation of SAS-4 and SAS-6 into centrioles. Like SAS-4 and SAS-6, alpha/beta-tubulin is stably incorporated into centrioles and is required for their duplication, gamma-tubulin, a specialized tubulin isoform implicated in microtubule nucleation also has a conserved role in centriole assembly. In the second aim, the ability to monitor the formation of centriolar substructures in living embryos will be used to identify the step(s) in the duplication cycle that require microtubule assembly and gamma-tubulin. A comparison of these results will indicate whether the primary role of gamma-tubulin is nucleation of centriolar microtubules. A mutation in the putative human homolog of SAS-4 was recently linked to autosomal recessive primary microcephaly (MCPH), a disorder associated with reduced brain size. Our work has also implicated a SAS-4-associated protein in a lethal fetal brain developmental disorder. In the final aim, a series of parallel experiments performed in C. elegans and mammalian cells will determine whether the mutations in these human disorders are associated with defects in the duplication of centrioles, or in one of their two critical functions, centrosome assembly or ciliogenesis. In humans, centrioles template the assembly of least 8 different types of cilia that propel mucus and fluids, coordinate developmental events, and move the egg and sperm during reproduction. In addition to contributing to our understanding of the regulation of brain size, characterizing how centrioles form and function during cell division and ciliogenesis will likely provide insight relevant to the diagnosis and treatment of the large spectrum of diseases associated with ciliary defects including: progressive blindness, infertility, polycystic kidney disease, Bardet- Biedel syndrome, hydrocephalus, and Kartagener's triad.
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