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 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 or1167 allele of the sas-6 gene, which encodes a downstream effector in the centriole duplication pathway, exhibits a meiosis specific defect. sas-6(or1167) is a temperature-sensitive missense mutation (Asp9Val). At the restrictive temperature, sas-6(or1167) animals exhibit a strong block in meiotic centriole duplication but very little to no effect on mitotic centriole duplication. Together these observations indicate that centrosome duplication is regulated differently during mitosis and meiosis. To understand these differences at a molecular level, we are working to identify factors that provide meiotic-specific regulation of centrosome duplication. To accomplish this we are isolating genetic suppressors of the sas-6(or1167) allele using the same approach that we successfully employed to identify regulators of zyg-1. Following chemical mutagenesis, we have identified 47 independent sas-6(or1167) strains that can grow for multiple generations at the restrictive temperature. So far only three suppressors exhibit dominance while 21 appear recessive. We have determined that one of the dominant suppressor mutations is intragenic and thus restores sas-6 function. 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) and deletion alleles of both genes exist. Both cdk-11 mutants exhibit a significant reduction in brood size but otherwise the phenotypes have not been well characterized. Using mutants alleles and RNAi we found that neither cdk-11 gene is required for centriole duplication in the embryo. To rule out genetic redundancy we simultaneously depleted both CDK-11 proteins by RNAi. Although animals subjected to double RNAi exhibited low fecundity and elevated embryonic lethality, such treatment did not produce a defect in centriole duplication. We also carried out a similar set of RNAi depletions in a sensitized genetic background (i.e. a strain partially compromised for the function of ZYG-1, the master regulator of centriole duplication) 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 down regulation of CDK-11 did not impact centriole duplication, we found that loss of either cdk-11.1 or cdk-11.2 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 and cdk-11.2 mutant hermaphrodites. Interestingly, only 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 genes in the late stages of gametogenesis and/or fertilization. Consistent with this, we found that cdk-11 mutants exhibit lower ovulation rates than wild-type controls. We are currently using a genetic approach to determine if the low fecundity can be attributed to defective eggs and/or sperm. The requirement for CDK-11 in reproduction might reflect a role in either the germ line itself or in the somatic gonad, which is required for normal fertility. To address 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 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.