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 number 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 co-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 marked by the presence of multipolar spindles. Interestingly, outside of the embryo, no other tissues appear to be affected by the ssna-1() mutation. Further, we found that while SSNA-1 is expressed in both the male and female germ lines, it is only required in the female germ line. That is, embryos sired by mutant males do not exhibit lethality while embryos sired by mutant mothers (regardless of the father’s genotype) exhibit strong embryonic lethality. Thus SSNA-1 might be a tissue specific regulator of centriole number. Druing the past year we have found that rather than functioning in centriole assembly, SSNA-1 acts to stabilize the centriole. In the absence of SSNA-1, centrioles form normally but break apart to produce centriole fragments that form extra spindle poles. This phenotype is somewhat reminiscent of that observed in embryos lacking the centriole stability protein SAS-1. Using ultrastructure expansion microscopy we have found that SSNA-1 and SAS-1 colocalize to a ring-like structure within the centriole lumen. Interestingly, both proteins also colocalize to a variable number of foci surrounding the PCM; these foci are reminiscent of centriole satellites observed in vertebrate cells but thought to be absent from invertebrates. Using classical genetic analysis, we further probed the function of SSNA-1 and found that ssna-1 and sas-1 genetically interact, further supporting a role for SSNA-1in stabilizing the centriole. Finally we found that SAS-1 is partially lost from centrioles that lack SSNA-1. Together these data indicate that SSNA-1 and SAS-1 function in close association to ensure the structural integrity of centrioles, a prerequisite for bipolar spindle assembly. While SSNA-1 and SAS-1 cooperate to stabilize the centriole, their contributions are not equal. Loss of ssna-1 results in only 70% embryonic lethality while mutations in sas-1 are fully embryonic lethal. Furthermore embryos lacking SSNA-1 only display centriole fragmentation and the associated multipolar-spindle phenotype, while those lacking SAS-1 display both multipolar (fragmentation) and monopolar spindles (a phenotype that presumably arises when whole nascent centrioles are lost). This suggests that SSNA-1 is only required for centriole stability post-assembly while SAS-1 is required both during and after centriole assembly. Consistent with this, we find that partial impairment of ZYG-1 enhances the embryonic lethal phenotype of ssna-1() mutants, leading to elevation of the multipolar phenotype as well as the appearance of monopolar spindles. Since the only known role of ZYG-1 is to promote formation of a stable cartwheel, these results suggests that SSNA-1, along with the cartwheel, stabilizes centrioles during assembly. Thus SSNA-1 contributes to centriole stability during assembly and is essental for stability following assembly. As part of a collaboration with Naoko Mizuno’s lab we also worked together to define the cryo-EM structure of SSNA-1and how its structure contributes to centriole stability. We find that SSNA-1 forms an anti- parallel coiled-coil dimer that self-associates via a triple stranded helical junction to form filaments. The filaments associate with microtubules in vitro through the helical junction, suggesting that the self-assembly of SSNA-1 creates hubs for microtubule binding. We also show that mutations that block self-assembly also fail to supports centriole stability and embryonic viability in vivo. In another project we are studying a role for the zinc-finger protein SZY-5 in controlling centriole assembly in the embryo. 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 and ultimately found that the miRNA pathway was not involved. We are now pursuing a second line of investigation. CGH-1 binds and stabilizes approximately 500 or so maternal mRNAs. Thus it seems likely that one or more of these maternal mRNAs plays an important role in regulating centriole assembly and is relevant to the mechanism of suppression. We are currently testing this hypothesis.