Over the past few years, we have identified and characterized a number of genes with novel roles in regulating centrosome size and centriole duplication. All such szy genes were identified in a screen for factors that genetically interact with the kinase ZYG-1, a conserved master regulator of centrosome duplication. Analysis of individual szy genes has led to the identification of several molecular pathways that control centriole duplication by controlling the expression levels of centriole assembly factors. Among our most recent findings, we have identified a novel pathway that negatively regulates centrosome duplication and relies upon the activity of protein phosphatase I (PP1). Loss of either the PP1- isoform GSP-1, or one of two highly conserved PP1 regulators (named I-2 and SDS-22), suppresses the centriole assembly defect of a zyg-1 hypomorphic mutation. This suggests that PP1 normally opposes the activity of ZYG-1, and accordingly we find a moderate increase in the level of ZYG-1 at centrosomes in embryos compromised for PP1 activity. Furthermore, we find that down regulation of PP1 activity results in a three- to five-fold increase in the total cellular levels of ZYG-1 protein, indicating that PP1 functions to limit expression of ZYG-1. As zyg-1 mRNA levels are unaffected by inhibition of PP1 activity, PP1 appears to act post-transcriptionally. Additionally, we have found that PP1 does not appear to regulate translation of zyg-1 through the 3 UTR. Our results indicate that PP1 acts post-translationally to control the stability of ZYG-1. Interestingly, we find that in a zyg-1(+) background, strong down regulation of either I-2, SDS-22, or both PP1 and PP1 results in the overproduction of centrioles, the formation of multipolar spindles and ultimately lethality. Using structured illumination microscopy (SIM) we have confirmed the centriole over-duplication defect and have found that more than one daughter forms next to each mother centriole. Thus, PP1 limits expression of ZYG- 1 to ensure the production of one and only one daughter centriole. These findings were published this past year in PLOS Genetics (Peel N., Iyer J., Naik A., Dougherty M. P., Decker M., O'Connell K. F., 2017 Protein Phosphatase 1 Down Regulates ZYG-1 Levels to Limit Centriole Duplication. PLoS Genet. 13: e1006543). Currently we are trying to identify the molecular target(s) of PP1. The most obvious candidate is ZYG-1, which contains a near-consensus PP1-docking motif. We have taken several approaches to determine if PP1 and ZYG-1 physically interact. These include co-immunoprecipitation (co-IP) experiments to determine if ZYG-1 and GSP-1 reside in a complex in vivo and mutating zyg-1 to ablate the docking motif and determine if this affects ZYG-1 protein levels. So far we have not been able to detect an interaction between ZYG-1 and PP1, I-2, or SDS-22. However, it is possible that co-IP experiments failed to detect an interaction because it is transient in nature. In another study, we have been characterizing a distinct regulator of centriole duplication. The chromodomain helicase CHD-1 is known to positively and negatively regulate transcription. In vertebrate cells, its known targets include genes encoding components of the centriole duplication pathway. Interestingly, CHD-1 co-precipitates with the ZYG-1 ortholog Plx4 in Xenopus egg extracts (Hatch et al. 2010 JCB 191: 721) suggesting the possibility that CHD-1 regulates centriole duplication. Consistent with this idea, we have found that RNAi of the worm chd-1 homolog suppresses the centriole duplication defect of the hypomorphic zyg-1(it25) mutant. We have also used CRISPR-based gene editing to construct a chd-1 null allele and have found that it also suppresses zyg-1(it25). Curiously, mutation of the ATPase domain of the endogenous protein leads to suppression, while mutation of the DNA-binding domain does not. Recently we have found that loss of chd-1 affects expression of certain components of the centriole assembly pathway. Specifically, chd-1 mutants show an increase in the levels of SPD-2 and SAS-6 proteins. Interestingly, spd-2 mRNA levels are also elevated in the mutant while sas-6 mRNA levels are unaffected. Our results indicate that CHD-1 regulates centriole duplication via an effect on transcription. During the past two years, we have been employing biochemical and biophysical approaches to characterize centriole duplication; specifically, we have been investigating how ZYG-1 might regulate (and be regulated by) downstream components of the centriole duplication pathway. SAS-5 and SAS-6 are coiled-coil-domain-containing proteins that form the structural scaffold of the centriole and require ZYG-1 for their incorporation into centrioles. These proteins are known to form dimers and higher order oligomers that are important for their function. However, the potential role of ZYG-1 in regulating the oligomeric state of these proteins has not been investigated. We have expressed full-length recombinant proteins in E. coli and purified them to near homogeneity. We have found that ZYG-1 and SAS-5 physically interact in vitro and that ZYG-1 is capable of phosphorylating SAS-5 in vitro. Further we have found that other centriole components (SAS-6 and SAS-4) can modulate ZYG-1 kinase activity. We mapped the phosphorylation sites in SAS-5 and used CRISPR-Cas9 gene editing to mutate these sites in the endogenous sas-5 gene. Unfortunately, we did not recover the desired mutants, as they appeared to be lethal as heterozygotes. We are now constructing RNAi-resistant transgenes carrying these mutations to determine what phenotypes might arise when the mutant transgenes are expressed in place of the endogenous sas-5 gene. Finally, we also are currently using analytical ultracentrifugation and other techniques to study how oligomerization of SAS-5 and SAS-6 in vitro might be regulated by ZYG-1. Together these experiments will allow us to better understand how these factors interact on a molecular level to build a nine-fold symmetric centriole
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