In most cell types, microtubules are organized by the centrosome, an organelle composed of a pair of centrioles surrounded by a matrix of pericentriolar material (PCM). During the cell cycle, the centrosome duplicates precisely once. This event is of critical importance to mitotic spindle assembly as it ensures that two centrosomes are available to form the poles of the bipolar spindle. Duplication involves splitting of the existing centriole pair followed by the synthesis of a new centriole next to each old centriole. As the cell progresses toward mitosis, the centrosome matures;that is, it accumulates PCM and by doing so, increases its microtubule nucleating capacity. Despite the importance of centrosome duplication and maturation, little is known of how these processes are regulated at a molecular level. In my laboratory, we are using the nematode Caenorhabditis elegans to study centrosome duplication and maturation. Specifically, our goals are to identify the factors that regulate these processes and to understand how they function on a molecular level. Over the past few years, we have identified and characterized novel regulators of centrosome size and duplication. All such szy genes were identified in a screen for factors that genetically interact with the kinase ZYG-1, a conserved upstream regulator of centrosome duplication. Most of our work has focused on SZY-20, a conserved RNA-binding protein that localizes to centrosomes and negatively regulates ZYG-1. We have demonstrated that SZY-20 limits centrosome size and that in its absence centrosomes are enlarged. These centrosomes possess elevated levels of ZYG-1 and PCM components such as SPD-2, SPD-5, and gamma-tubulin. Further, we have found that these enlarged centrosomes nucleate more microtubules than their wild-type counterparts and that the enlarged centrosome phenotype is associated with defects in microtubule-dependent processes. Of particular significance we discovered that two centrosome duplication factors, ZYG-1 and SAS-6 play positive roles in defining centrosome size. While disruption of the SZY-20/ZYG-1-mediated size control mechanism affects PCM levels, centriole structure is unperturbed, indicating that the role of ZYG-1 in controlling centrosome size is separable from its role in centriole duplication. Analysis of two other szy genes has revealed a role for protein phosphatase I (PP1) in negatively regulating centrosome duplication. Loss of either the PP1 isoform GSP-1 or one of two PP1 regulators (named I-2 and SDS-22) suppresses the centriole assembly defect of a zyg-1 hypomorphic mutation. While this suggests that PP1 normally opposes the activity of ZYG-1, the level of ZYG-1 at centrosomes is unaffected by loss of PP1 activity. Instead, we find that the centrosome level of SPD-2, another factor required for centriole assembly, is elevated in PP1-deficient cells. A number of kinases are known to promote accumulation of SPD-2 at centrosomes. These include ZYG-1, PLK-1, AIR-1, and cdk11. PP1 might normally oppose the activity of one or more of these kinases, and thus suppression of zyg-1 by PP1 depletion might be due to an effective increase in the activity of one of these kinases. So far our work suggests that PP1 is not opposing ZYG-1 or cdkll and experiments are underway to test PLK-1 and AIR-1. Our work has also uncovered a role for yet another kinase in the centriole assembly pathway. The kinase PAR-1 is a conserved regulator of cell polarity and localizes to the cell cortex and cytoplasm where it regulates the distribution of downstream cell fate determinants. PAR-1 also localizes to centrosomes but role for PAR-1 at centrosomes has not been described. We have found that the szy-11(bs22) mutation is a rare gain-of-function allele of the par-1 gene and can suppress the centriole duplication defect of a zyg-1 mutant. In contrast, depletion of PAR-1 by RNAi can enhance the zyg-1 centriole duplication defect. These results suggest that PAR-1 normally functions alongside ZYG-1 to promote centriole assembly. Although we have yet to determine how PAR-1 promotes centriole duplication, our work indicates that PAR-1 may be one of the kinases that are opposed by PP1. Further work should provide important new insights into how centriole assembly is regulated by a growing network of kinases and phosphatases. We are also trying to understand how the cell limits centriole duplication to a single round per cell cycle. Deregulation of centrosome duplication causes the appearance of supernumerary centrosomes, which are a hallmark of many cancer cells and can contribute to tumorigenesis. Ectopic expression of the ZYG-1-related kinase Plk4 in vertebrate cells causes the formation of extra centrosomes, and aberrant Plk4 expression levels are associated with cancer. Data from Drosophila and human cells show that Plk4 levels are regulated by the SCF-bTrCP ubiquitin ligase and proteasomal degradation. bTrCP serves as the substrate recognition component of this ubiquitin ligase. However, human Plk4 is still degraded in the absence of bTrCP recognition, suggesting the existence of additional regulatory mechanisms. We have found that in C. elegans, ZYG-1 levels are also regulated by proteasomal degradation and that the SCF ubiquitin ligase complex is required for this degradation. Uniquely, we find that both the bTrCP homolog, LIN-23 and the related F-box protein SEL-10 function redundantly in SCF-mediated ZYG-1 degradation. SEL-10 is the homolog of FBW7, a human gene that is frequently mutated in cancer. Our findings therefore suggest that mutation of FBW7 may increase the risk of cancer by deregulating normal centrosome duplication.

Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
2012
Total Cost
$443,402
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Miller, Jacqueline G; Liu, Yan; Williams, Christopher W et al. (2016) The E2F-DP1 Transcription Factor Complex Regulates Centriole Duplication in Caenorhabditis elegans. G3 (Bethesda) 6:709-20
O'Connell, Kevin F; Golden, Andy (2014) Confocal imaging of the microtubule cytoskeleton in C. elegans embryos and germ cells. Methods Mol Biol 1075:257-72
Peel, Nina; Dougherty, Michael; Goeres, Jacqueline et al. (2012) The C. elegans F-box proteins LIN-23 and SEL-10 antagonize centrosome duplication by regulating ZYG-1 levels. J Cell Sci 125:3535-44
Decker, Markus; Jaensch, Steffen; Pozniakovsky, Andrei et al. (2011) Limiting amounts of centrosome material set centrosome size in C. elegans embryos. Curr Biol 21:1259-67
Peters, Nathaniel; Perez, Dahlia E; Song, Mi Hye et al. (2010) Control of mitotic and meiotic centriole duplication by the Plk4-related kinase ZYG-1. J Cell Sci 123:795-805
Kumfer, Kraig T; Cook, Steven J; Squirrell, Jayne M et al. (2010) CGEF-1 and CHIN-1 regulate CDC-42 activity during asymmetric division in the Caenorhabditis elegans embryo. Mol Biol Cell 21:266-77
Song, Mi Hye; Aravind, L; Muller-Reichert, Thomas et al. (2008) The conserved protein SZY-20 opposes the Plk4-related kinase ZYG-1 to limit centrosome size. Dev Cell 15:901-12
Song, Mi Hye; Miliaras, Nicholas B; Peel, Nina et al. (2008) Centrioles: some self-assembly required. Curr Opin Cell Biol 20:688-93