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 paternal (male meiotic) centriole duplication. At the restrictive temperature, this sas-6 mutant produces sperm that are only capable of directing formation of a monopolar spindle during the first embryonic division. Prior studies have shown that such a phenotype arises as a result of male meiotic centriole failure leading to sperm with a single centriole, rather than a centriole pair. In contrast maternally-controlled mitotic centriole duplication is only blocked 60 percent of the time. Together these observations suggest that different cell types might utilize different mechanisms for regulating centriole number. 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 core component of the centriole scaffold. The sas-6(or1167) mutation changes an aspartate residue in the N-terminal head region to valine (SAS-6-D9V). 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. Furthermore, we have purified wild-type and mutant recombinant protein and found that the mutant protein is less soluble than the wild type and undergoes auto-cleavage in vitro. Thus the D9V substitution likely affects folding and long-term stability of SAS-6. Recently, we have found that many of the monopolar spindles formed in the sas-6(or1167) mutant lack detectable centriole proteins, indicating a complete absence of centrioles. This phenotype is not consistent with a simple duplication defect but rather suggests loss of centriole stability. Consistent with this, we have analyzed intact male germ lines and found that centriole markers are present in most cells prior to meiosis but are gradually lost as sperm form. In collaboration with Thomas Muller-Reichert of MTC-Dresden, we are analyzing centriole structure in mutant sperm. So far we have found that mutant sperm contain a single structurallyabnormal centriole. Thus the sas-6(or1167) appears to disrupt centriole integrity. 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 number. In conjunction with the NIDDK Genomics Core Facility, we have begun whole genome sequencing of twelve of the strongest suppressors. We have identified one suppressor as an allele of zyg-1. The zyg-1(bs84) mutation is in the SAS-6-binding domain suggesting that ZYG-1 might regulate SAS-6 stability. We are currently exploring this possibility. In a related 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. However, a cdk-11.1; cdk-11.2 double null mutant displayed a synthetic 100% penetrant larval lethal phenotype, indicating that the two genes are partially redundant. Using a combination of mutant 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. Recently we found that loss of CDK-11.1 results in a failure of MAP kinase signaling in the germ line. MAP kinase is required for the production and maturation of oocytes. Thus CDK-11 might regulate oogenesis via MAP kinase signaling.