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 dis-engagement of the existing centriole pair followed by the synthesis of one new (daughter) centriole next to each pre-existing (mother) 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 a number of genes encoding 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. 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. Over the past year we have been focused on two pathways of particular interest, one of which operates at a post-transcriptional level and a second that operates at a transcriptional level. The first pathway involves the activity of protein phosphatase I (PP1) in negatively regulating centrosome duplication. Loss of either the PP1-βisoform 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 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 to regulate zyg-1. Interestingly, we find that in a zyg-1(+) background, strong down regulation of either I-2 or SDS-22 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 found that more than one daughter forms next to each mother centriole. Currently we are trying to identify the molecular target(s) of PP1. The most obvious candidate is ZYG-1, which contains a consensus PP1-docking motif. We are taking several approaches to determine if PP1 and ZYG-1 physically interact. These include co-immunoprecipitation experiments to determine if ZYG-1 and GSP-1 reside in a complex in vivo and mutating zyg-1 using CRISPR/cas9-mediated genome editing to ablate the docking motif and determine if this affects ZYG-1 protein levels. We have also used mass spectrometry to identify additional proteins that co-precipitate with SDS-22 and I-2 and are screening these candidate interactors for a role in regulating centriole duplication. The second pathway of interest regulates centriole duplication at a transcriptional level. This is an important yet overlooked area of investigation as most studies have focused on post-transcriptional mechanisms of regulation. We have found that in the worm, the heterodimeric transcription factor E2F-DP1 represses centrosome duplication. Specifically we find that loss of E2F or DP1 activity can suppress a zyg-1 hypomorphic allele. Accordingly, conserved binding sites for this transcription factor are found in the promoter regions of most of the centriole duplication genes including zyg-1 and sas-6, and recent ChiP-on-Chip analysis demonstrates that these sites are bound in vivo by E2F-DP1 (Kudron et al. 2013 Genome Biol. 14: R5). We have performed qRT-PCR analysis of transcript levels in wild-type and dpl-1 mutants and find that E2F-DP1 is likely to play a positive role in regulating transcription of centriole duplication genes. Consistent with this, deletion of E2F-binding site in the promoter of ZYG-1 abolishes expression. Despite our finding that E2F-DP1 plays a positive role in regulating expression of centriole duplication genes including SAS-6, we find that loss of E2F-DP1 specifically results in an increase in the level of SAS-6 protein. This seemingly contradictory result can be explained if E2F-DP1 also positively regulates the expression of a gene that down regulates SAS-6 protein levels. Our data thus favors a model whereby E2F-DP1 through its role in transcription sets the balance between positive and negative regulators of centriole duplication and that a partial loss of E2F-DP1 tips the balance in favor of the positive regulators. Finally, we have also begun to characterize another regulator of centriole duplication. The chromodomain helicase CHD-1 is known to positively and negatively regulate transcription. Among its known targets are genes encoding components of the centriole duplication pathway. Recently, CHD-1 was found to be co-precipitated with the presumptive ZYG-1 homolog Plx4 in Xenopus egg extracts (Hatch et al. 2010 JCB 191: 721). We have found that depletion of the worm chd-1 homolog suppresses the centriole duplication defect of the zyg-1(it25) mutant. A deletion in the chd-1 gene can also suppress zyg-1(it25) indicating that chd-1 negatively regulates centriole duplication. While loss of chd-1 function does not affect ZYG-1 or SAS-6 protein levels, the SAS-6 protein of chd-1 mutant worms exhibits altered mobility by SDS-PAGE indicating an altered post-translational modification. We are currently trying to identify this modification and how it might be regulated by CHD-1.
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