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 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. To gain further insight into the mechanism of SZY-20 action, we utilized a proteomics approach to identify proteins that specifically interact with SZY-20. We have identified a large number of proteins that reproducibly co-immunoprecipitate with SZY-20. Among these interactors are known RNA-binding proteins, and regulators of the microtubule cytoskeleton. To determine which of these proteins may play an important role in regulating the centrosome, we used RNAi to deplete each factor from otherwise wild-type animals and monitored centrosome behavior. Depletion of several factors disrupted various centrosome-associated processes. In particular, depletion of the catalytic subunit of protein phosphatase 2A (PP2A) produced a centrosome duplication defect identical to that of ZYG-1. Similarly, depletion of either PAA-1, the PP2A scaffolding subunit, or SUR-6, a PP2A regulatory subunit, resulted in a centrosome duplication phenotype. Using classical genetics, we have found that ZYG-1 and PP2A exhibit a strong genetic interaction, indicating that these two factors function closely together to regulate centriole duplication. In addition, we find that PP2A and the centriole duplication factor SAS-5 also exhibit a robust genetic interaction. Consistent with our genetic results, we find that PP2A physically interacts with SAS-5 in vivo, and is required to maintain proper levels of ZYG-1 and SAS-5. Finally, we have found that PP2A-SUR-6 is required for recruitment of SAS-5 to sites of centriole assembly. Overall our results indicate that PP2A-SUR-6 functions to maintain the levels of ZYG-1 and SAS-5.. Since ZYG-1 is required for the recruitment of SAS-5, the reduced levels of ZYG-1 and SAS-5 in PP2A-SUR-6-deficient embryos likely accounts for the failure of SAS-5 to accumulate at sites of centriole assembly resulting in the block in centriole duplication. While our data demonstrate that PP2A plays a positive role in centriole assembly, we have also found that protein phosphatase I (PP1) plays a negative role. Loss of either the PP1 isoform GSP-1 or one of two PP1 regulators (named I-2 and T09a5.9) 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. We are currently exploring this possiblity. Our work should provide important new insights into how centriole assembly is regulated by a balance 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 SCFSlimb/bTrCP ubiquitin ligase and proteasomal degradation. However, human Plk4 is still degraded in the absence of βTrCP 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 the F-box protein SEL-10, rather than the Slimb/bTrCP homolog, LIN-23, is required for 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
9
Fiscal Year
2011
Total Cost
$597,804
Indirect Cost
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Peel, Nina; Iyer, Jyoti; Naik, Anar et al. (2017) Protein Phosphatase 1 Down Regulates ZYG-1 Levels to Limit Centriole Duplication. PLoS Genet 13:e1006543
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
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