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. 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. During the past few years, we have been characterizing the effects of the sas-6(or1167) mutation on centriole assembly and stability. SAS-6 is a coiled-coil domain protein and a core component of the centriole. It is thought that centriole biogenesis involves the self-assembly of SAS-6 molecules into a nine-fold symmetric scaffold around which the centriole is constructed. In this model, the mother centriole only serves to define the site of centriole synthesis but does not play an active role in formation of the daughter. 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. Importantly, we have found that the D9V form of SAS-6 leads to either failure of daughter centriole assembly or formation of a replication-compromised daughter. Recently, we have found that many of the monopolar spindles formed in sas-6(or1167) embryos lack core centriole components such as SAS-4 and SAS-6, indicating that the structure of the centriole is severely compromised. Similarly, in sas-6(or1167) male germ lines, we find that centriole markers are present in most cells prior to meiosis but are gradually lost as centrioles age. In collaboration with Thomas Muller-Reichert of MTC-Dresden, we have found by electron microscopy that sas-6(or1167) centrioles (termed D9V centrioles for convenience) are indeed abnormal in structure, and in some instances, appear to lack the nine-fold symmetry characteristic of normal centrioles. Thus, it appears that the sas-6(or1167) mutation not only impairs centriole assembly but also affects the stability of those centrioles that do assemble. Why the stability defect is more apparent in the male germ line is not yet clear. 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. Loss of SZY-5 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. Most relevantly, a recent publication has reported that the role of CGH-1 in miRNA function is casein kinase 2 (CK2) dependent, while another publication showed that depletion of CK2 suppresses a zyg-1 mutant. Strengthening the link between SZY-5 and CGH-1, we recently found that CGH-1 is poorly expressed in szy-5 mutants and that depletion of CGH-1 can suppress the centriole duplication defect of zyg-1(it25) mutants. Altogether, these results suggest that suppression of zyg-1(it25) upon loss of either SZY-5 or CGH-1 arises from a defect in miRNA function. We are currently exploring this possibility. Finally, we are working in collaboration with Naoko Mizuno (NHLBI) to study SSNA-1 (Sjoegren Syndrome Nuclear Autoantigen 1), 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. To investigate the function of SSNA-1 in an intact organism we 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(delta) 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 involves the overexpression of centriole assembly factors such as SAS-5 and SAS-6. However, we have found that such factors are not overexpressed in the ssna-1(delta) 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 the surrounding PCM. We are currently testing to see if SSNA-1 might use its microtubule-binding and remodeling function to directly suppress the formation of excess daughter centrioles.