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. We expect that in some of these suppressor strains, the sas-6(or1167) mutation has reverted back to wild-type while in others a compensating mutation in the sas-6 gene (intragenic suppressor) has arisen. For our purposes, the most informative class will be those that carry a mutation in a gene other than sas-6 (extragenic suppressors), as these can be used to identify potentially important meiosis-specific regulators of centriole duplication. So that we can concentrate our efforts on extragenic suppressors we are sequencing the sas-6 gene in each of the suppressors. All eight of those suppressor strains so far sequenced contain the original or1167 mutation but no other sas-6 mutation. Thus our screen is likely to have identified a large number of extragenic mutations. In conjunction with the NIDDK Genomics Core Facility, we plan to molecularly identify suppressors by whole genome sequencing. Once identified suppressors will be studied individually to understand how they may contribute to the regulation of meiotic centriole assembly. In a second approach to identify tissue-specific regulators of centriole duplication, we are currently working to develop the technique of BioID for use in C. elegans. BioID is a method for selectively tagging interacting or proximal proteins with biotin. Developed by the Roux lab for use in tissue culture (Roux et al. 2012, J Cell Biol. 196:801-10), this method uses a promiscuous biotin ligase (birA*) fused to a protein of interest. birA* can activate biotin which then diffuses from the active site and biotinylates neighboring proteins. These biotinylated candidate proteins can then be affinity purified and identified by mass spectroscopy. We are constructing genes encoding birA* fusion proteins to test this method in worms. So far we have found that unlike tissue culture cells, worms contain a high background of endogenous biotinylated proteins. Several approaches exist to reduce or eliminate this background and we are currently trying these. Once we minimize the background we will express and test fusions of birA* to various centrosome and centriole proteins. The initial constructs will be expressed under control of the native promoters and will therefore be expressed in all tissues where the endogenous centrosome protein is expressed. If the technique is successful, we will then carry out BioID using fusion proteins expressed under control of tissue-specific promoters. This will allow us to identify tissuespecific patterns of centrosome composition.

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