Developmental Regulation of Centrosome Duplication

O'Connell, Kevin

Abstract

Centrosomes are the primary microtubule-organizing centers (MTOCs) in most cells and consist of a pair of centrioles wrapped within a cloud of pericentriolar material (PCM). In Caenorhabditis elegans, the kinase ZYG-1 is essential for the duplication of centrioles. Embryos lacking maternal ZYG-1 activity fail to duplicate the paternally contributed centriole pair, and are thus unable to form bipolar spindles following first division. In contrast, loss of paternal ZYG-1 activity results in duplication failure during male meiosis, and the production of sperm with a single centriole. These sperm can still fertilize eggs but the resulting embryos assemble a monopolar rather than bipolar spindle at first division. These results demonstrate that ZYG-1 is required for centriole duplication during both the mitotic divisions of the embryo and the meiotic division of spermatocytes. Although ZYG-1 and other components of the centriole assembly pathway are absolutely required for centriole duplication during mitosis and meiosis, some recent data indicates that these factors are regulated differently during the two modes of division. We have found that small truncations of the c-terminus of ZYG-1 block centriole duplication during mitosis but drive the over-duplication of centrioles during meiosis. The behavior of these truncated forms of ZYG-1 seems to reflect their ability to localize to centrioles; the mutant proteins can accumulate at the meiotic centrioles of spermatocytes but are unable to localize to the mitotic centrioles of embryos. Similarly, we have found that the temperature-sensitive sas-6(or1167) mutation appears to mostly affect meiotic centriole duplication. At the restrictive temperature, meiotic centriole duplication is completely blocked while mitotic centriole duplication is only blocked 60 percent of the time. Together these observations indicate that centrosome duplication is regulated differently during mitosis and meiosis. During the past year we have begun a detailed characterization of the effects of the sas-6(or1167) mutation. SAS-6 is a coiled-coil domain protein and a component of the centriole scaffold. The sas-6(or1167) mutation changes an aspartate residue in the N-terminal head region to valine. Since the globular head of SAS-6 mediates the oligomerization needed to form a centriole scaffold, the or1167 mutation might affect the packing of the protein within the scaffold. This might lead to either a complete failure in centriole duplication or formation of an unstable centriole. Immunoblotting experiments reveal that the protein encoded by sas-6(or1167) is expressed at a lower level than wild-type SAS-6. This defect is most severe at restrictive temperature, suggesting that the temperature-sensitive nature of this allele is due to an unstable protein. As part of this project, we also are isolating genetic suppressors of the sas-6(or1167) allele using the same approach that we successfully employed to identify regulators of zyg-1. So far we have identified 41 independent sas-6(or1167) strains that can grow for multiple generations at the restrictive temperature. Six of the suppressors exhibit dominance while 23 appear recessive. One of the dominant suppressor mutations was found to be intragenic and results in a second missense mutation in the head region of SAS-6. It is likely that most, if not all, of the remaining suppressors carry a mutation in a gene other than sas-6 (extragenic suppressors); these can be used to identify potentially important meiosis-specific regulators of centriole duplication. In conjunction with the NIDDK Genomics Core Facility, we have begun whole genome sequencing of twelve of the strongest suppressors. Once a suppressor is molecularly identified, it will be studied individually to understand how it might contribute to the regulation of meiotic centriole assembly. As part of this project, we have begun to study the function of the cyclin-dependent kinase CDK-11 in the germ line. CDK-11 is a conserved kinase with established roles in transcription, microtubule nucleation, and apoptosis. Recently a published report demonstrated an essential role for human CDK-11 in centriole duplication in somatic cells (Franck et al . (2011). PLoS ONE, 6(1), e14600). Our initial objective was to determine if CDK-11 functioned in a similar capacity in the C. elegans embryo and if possible to further dissect its role in the centriole assembly pathway. C. elegans possesses two cdk-11 genes (cdk-11.1 and cdk-11.2). A deletion (null) allele of cdk11.1 was available. As a null cdk-11.2 allele was not available we used CRISPR technology to cleanly delete the cdk-11.2 gene. Deletion of cdk-11.2 did not produce an observable phenotype while deletion of cdk-11.1 caused a significant reduction in brood size. Using a combination of mutants alleles and RNAi we did not find evidence that the cdk-11 genes are required for centriole duplication in the embryo. We also carried out a set of RNAi depletions in a sensitized genetic background (i.e. a strain partially compromised for the function of ZYG-1) and did not detect an effect on centriole duplication. Our results indicate that CDK-11 does not play an important role in centriole duplication in the worm embryo and suggests that this particular function of CDK-11 is either not conserved or that CDK-11 function is tissue-specific. While CDK-11 does not appear important for centriole duplication, we found that loss of CDK-11.1 resulted in a reduction in fecundity and a small but significant level of embryonic lethality. To understand the underlying cause of the low fecundity we examined the germ lines of cdk-11.1 hermaphrodites. Interestingly, the cdk-11.1 mutant displayed an obvious defect that was localized to the most proximal region of the germ line and involved variable morphological defects of oocytes and sperm. This finding suggests a role for the cdk-11.1 in the late stages of gametogenesis and/or fertilization. We performed genetic analysis and found that cdk-11.1 is required in both the male and female germ lines. Interestingly, we found that while cdk-11.1 mutant sperm can crawl and respond normally to signals from wild-type oocytes, they do not appear to signal ovulation and are incapable of fertilization. Similarly, mutant oocytes appear incapable of signaling wild-type sperm. Thus CDK-11.2 might be required for gametes to signal their partner during fertilization. To determine where CDK-11 might be functioning we sought to determine where it is expressed. We therefore made transcriptional/translational reporter constructs and found that both cdk-11 homologs are expressed throughout the entire germ line and also in the somatic cells of the gonad. We then produced an antibody specific for CDK-11.2 (production of a CDK-11.1 antibody failed) and found that endogenous CDK-11.2 is enriched in the meiotic cells of the germ line and in the nuclei of the gonadal sheath cells. We are currently designing experiments to test whether the low fecundity observed in cdk-11.1 mutants arises from defects in the germ line and/or soma. In summary, our evidence indicates that CDK-11 is expressed in both the germ line and somatic gonad and is required for the production of fertilization-competent gametes.