Developmental Regulation of Centrosome Duplication

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Centrosomes are the primary microtubule-organizing centers (MTOCs) in most cells and consist of a pair of centrioles within a cloud of pericentriolar material (PCM). During mitosis, each centrosome establishes one pole of the bipolar spindle. 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 data indicate that these factors are regulated differently during the two modes of division. For instance, 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 differentially affect meiotic and mitotic centriole duplication. At the restrictive temperature, the sas-6(or1167) mutant strongly blocks male meiotic centriole duplication leading to an invariant monopolar spindle defect during the first embryonic division. 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. One factor that seems to function in a tissue-specific manner to control centriole assembly is the microtubule remodeling factor SSNA-1 ((Sjoegren Syndrome Nuclear Autoantigen 1). SSNA-1 is a small coiled-coil protein that has been shown to polymerize along the wall of microtubules to promote microtubule branching. When overexpressed in cultured neurons SSNA-1 promotes axon branching in a manner dependent on its microtubule remodeling activity. In collaboration with Naoko Mizuno (NHLBI) we are investigating the function of SSNA-1 in an intact organism and have used CRISPR to knock out the worm ortholog of SSNA-1. We find that while the gene is not absolutely essential, worms homozygous for the SSNA-1 deletion allele exhibit high levels of embryonic lethality and morphology defects such as vulval defects and a failure of the posterior to form. Examination of ssna-1() embryos revealed the presence of multipolar spindles that appear to arise as a consequence of centriole overduplication. Interestingly, these extra spindle poles generally arise only after the first two cell cycles suggesting that SSNA-1 function might be restricted to the later stages of embryogenesis. One potential mechanism to drive the overproduction of centrioles is to overexpress centriole assembly factors such as SAS-5 and SAS-6. However, we have found that such factors are not overexpressed in the ssna-1() mutant. This suggests a more direct role for SSNA-1 in controlling centriole assembly. Consistent with this, we find that SSNA-1 localizes to centrioles and to a variable number of foci surrounding the PCM. This past year we have found that deletion of the ssna-1 gene affects the localization of centriole assembly factors. Specifically, we find that the levels of ZYG-1 are reduced at centrioles, while the levels of SAS-6 are elevated beginning at the two-cell stage. Although the significance of the reduction in ZYG-1 levels at the centrosome is unclear, the increased levels of SAS-6 might explain the multipolar spindle phenotype. Using classical genetic analysis, we have further probed the function of SSNA-1. Importantly we found that zyg-1 and ssna-1 genetically interact, as a zyg-1(it25); ssna-1() double mutant exhibits significantly more embryonic lethality than expected. Our preliminary data indicates that that a partial loss of zyg-1 function enhances the multipolar spindle phenotype of the ssna-1 mutant. We are currently trying to understand the basis for this unexpected result. Finally, we found that while SSNA-1 is expressed in both the male and female germ-lines, it is only required in the female germline. That is embryos sired by mutant males do not exhibit lethality while embryos sired by mutant mothers (regardless of the fathers genotype) exhibit strong embryonic lethality. Thus SSNA-1 might be a tissue specific regulator of centriole assembly. MicroRNAs (miRNAs) are small RNA molecules that posttranscriptionally repress gene expression by binding to complimentary sequences in the 3 untranslated region of target mRNAs. A number or miRNAs are known to be expressed in the C. elegans female germline and embryo but the targets of these miRNAs and the processes they regulate are not well characterized. We are currently studying a potential role for the zinc-finger protein SZY-5 in miRNA function. SZY-5 is a maternally-expressed protein whose loss suppresses the centrosome duplication defect of a zyg-1(it25) mutant and results in cytological defects identical to those observed in cgh-1 mutants. CGH-1 is a conserved germ line helicase that functions in numerous processes including processing body formation and miRNA function. We have found that SZY-5 is required for normal expression of CGH-1; in szy-5 mutants, both CGH-1 protein and mRNA levels are significantly reduced relative to the wild type, indicating that SZY-5 normally functions to control the production or stability of cgh-1 mRNA. Further, we find that loss of cgh-1 (like loss of szy-5) suppresses the centriole assembly defect of a zyg-1(it25) mutant. This indicates that CHG-1 functions downstream of SZY-5 to regulate centriole assembly. To determine if CGH-1 regulates centriole assembly though its role in the miRNA pathway, we used a mutation in the pash-1 gene to block miRNA biogenesis in the zyg-1(it25) mutant. Consistent with our hypothesis, loss of miRNA production strongly restored centriole assembly. We are now trying to identify the specific miRNAs that are involved and their target in the centriole assembly pathway. Finally, we have initiated a new project to understand how centriole stability might be regulated in a tissue-specific manner. SAS-1 is a centriole-associated factor that is required specifically for centriole stability in the embryo. That is, loss of SAS-1 has no effect on centrioles in the male germline but upon being donated to the embryo at fertilization, the sperm-derived centrioles become unstable. How SAS-1 functions to stabilize centrioles is unknown. To begin to address this question we have initiated a screen for mutations that restore centriole stability to a temperature-sensitive sas-1 loss-of-function mutant. We have identified 13 suppressors that allow the sas-1 mutant to grow at restrictive temperature. We are currently preparing genomic preps for deep sequencing and mapping of the mutant alleles. We hope to be able to identify additional genes that function with SAS-1 and to study these newly discovered genes to understand the molecular basis of centriole stability.