In most cell types, microtubules are organized by the centrosome, an organelle composed of an orthogonal 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"""""""" or gradually acquires increased levels of microtubule nucleating capacity and PCM. 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 replication. To gain further insight into the mechanism of SZY-20 action, we have 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. 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 centrosome duplication. We are continuing to characterize the role of PP2A in centrosome duplication as well as its interaction with SZY-20. This past year we have also continued our studies of SZY-5, another negative regulator of ZYG-1. SZY-5 encodes a zinc-finger protein, and like SZY-20, negatively regulates centrosome size. However, SZY-5 appears to function in a manner distinct from that of SZY-20. Unlike SZY-20, SZY-5 does not localize to centrosomes and does not appear to regulate the centrosome-levels of ZYG-1. SZY-5 is found primarily in small cytoplasmic puncta, a distribution reminiscent of processing bodies (P-bodies), which are sites where mRNA stability and translation are controlled. We have postulated that SZY-5 might control the expression of one or more centrosome duplication factors and have used RT-PCR and quantitative immunostaining to address this possibility. We have found that SZY-5 does not significantly affect the mRNA levels of known centrosome duplication factors. However loss of SZY-5 leads to an increase in the levels of SPD-2 at the centrosome. Our results so far indicate that no other centrosome proteins are affected by loss of SZY-5. We have also found that embryos lacking SZY-5 display defects in osmotic integrity, cell cycle progression, polar body extrusion, cytokinesis, and anaphase spindle positioning. As some of the processes affected by the szy-5 mutation involve centrosome function, we are examining centrosome structure and function in the mutant. Finally, we are initiating a proteomics approach to study SZY-5, as the identification of interacting factors should help us understand the role of SZY-5 in centrosome duplication. We have also made significant progress in our characterization of another szy gene. Mutation of the szy-2 gene suppresses a partial loss of ZYG-1 activity. We cloned szy-2 and found that it encodes a regulatory subunit of protein phosphatase 1 (PP1) named I-2. Our genetic analysis indicates that the szy-2 mutation reduces the activity of I-2. Preliminary results indicate that this leads to a down-regulation of PP1 activity, suggesting that I-2 normally opposes ZYG-1 by modulating PP1 activity. Loss of I-2 mediated regulation does not affect the level of ZYG-1 at centrosomes, suggesting that it acts downstream of ZYG-1 in the centrosome duplication pathway. Since ZYG-1 is a kinase, PP1 may oppose ZYG-1 activity by acting upon one or more of its substrates. In addition to its interaction with ZYG-1, we find that I-2 is required for proper chromosome segregation.

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Schwarz, Anna; Sankaralingam, Prabhu; O'Connell, Kevin F et al. (2018) Revisiting Centrioles in Nematodes-Historic Findings and Current Topics. Cells 7:
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
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
Song, Mi Hye; Liu, Yan; Anderson, D Eric et al. (2011) Protein phosphatase 2A-SUR-6/B55 regulates centriole duplication in C. elegans by controlling the levels of centriole assembly factors. Dev Cell 20:563-71
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

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