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 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 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. Amongst the regulators we have so far characterized is SZY-20, a conserved RNA-binding protein that localizes to centrosomes where it inhibits the accumulation of ZYG-1 and a number of other centrosome components. Loss of SZY-20 activity results in a ZYG-1-dependent increase in PCM size and microtubule nucleating capacity. Thus SZY-20 acts in a negative manner to control centrosome size. 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. 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 loss 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, loss of 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 found that more than one daughter forms next to each mother centriole. Currently we are trying to identify the molecular target(s) of PP1. To this end we have used mass spectrometry to identify proteins that co-precipitate with SDS-22 and I-2, and are now focusing on one of these interacting proteins: CSTL-2, a worm protein related to an RNA-binding that promotes the formation of a poly-A tail at the 3-end of messenger RNA. Loss of CSTL-2, like loss of PP1, suppresses the centrosome duplication defect of a zyg-1 mutant. Our data support a model in which PP1 acts through CSTL-2 to limit expression of ZYG-1 and thereby prevent the over-duplication of centrioles. As illustrated by our work on PP1, understanding how a cell limits centriole duplication to a single round per cell cycle is of great importance as the presence of supernumerary centrioles can disrupt spindle structure and such defects have been linked to tumorigenesis. Over-expression of the ZYG-1-related kinase Plk4 in vertebrate cells causes the formation of extra centrioles, 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. Additionally, we are also investigating how centrosome duplication might be regulated 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). Surprisingly however, our quantitative PCR analysis has not revealed a role for E2F-DP1 in regulating transcription of centriole duplication genes, yet we have found that loss of DP1 results in an increase in the level of SAS-6 protein. Our data thus favors a model whereby E2F-DP1 controls expression of some as yet unknown factor that in turn negatively regulates SAS-6 protein levels. Finally, we have begun to analyze the molecular interactions that underlie the duplication and maturation of centrosomes. ZYG-1 requires the coiled-coil protein SPD-2 for localization to centrioles while SPD-2 requires ZYG-1 to localize properly to the PCM. To investigate how these two proteins regulate each other, we have expressed and purified these proteins from bacteria. We have found that ZYG-1 and SPD-2 physically interact and have mapped the sites of interaction in the two proteins. Furthermore we have found that SPD-2 is a substrate of ZYG-1 in an in vitro kinase reaction and have evidence that ZYG-1 mediated phosphorylation of SPD-2 can be stimulated by addition of the centriole duplication factor SAS-6 to the in vitro reaction. Currently we are trying to identify the minimal regions required for the SPD-2-ZYG-1 interaction and any amino acid residues that play a critical role. We are also working to identify the residues in SPD-2 that are phosphorylated by ZYG-1. Using this information, we will create transgenic worm lines that express the various mutant versions of SPD-2 and ZYG-1 in place of the endogenous genes. We will then analyze such strains to determine if such mutations affect centriole duplication and/or centrosome maturation.
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