Centrosome Maturation and Duplication in the C. elegans Embryo

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Over the past few years, we have identified and characterized a number of genes with novel roles in regulating centrosome size and centriole duplication. While we have a general understanding of how these genes function, we lack detailed knowledge of the molecular interactions that take place during centriole and centrosome assembly. One area of active investigation involves understanding how phosphorylation controls centriole assembly. The kinase ZYG-1 is considered the master regulator of centriole assembly, but how it functions is poorly understood. SAS-5 and SAS-6 are coiled-coil-domain-containing proteins that form the structural scaffold of the centriole and require ZYG-1 for their incorporation into nascent centrioles. These proteins are known to form dimers and higher order oligomers that are important for their function. However, the potential role of ZYG-1 in regulating the oligomeric state and intermolecular interactions of these proteins have not been investigated. We have expressed full-length recombinant proteins in E. coli and have purified them to near homogeneity. We have found that ZYG-1 and SAS-5 physically interact in vitro and that ZYG-1 is capable of phosphorylating SAS-5 in vitro. Using mass spectrometry, we find that ZYG-1 phosphorylates SAS-5 on a number of highly conserved serine and threonine residues. Of particular interest, four of these phosphorylated residues are in a region we have shown binds to ZYG-1; this suggests that ZYG-1 might modulate its own binding to SAS-5. To address this, we used site directed mutagenesis to create a series of non-phosphorylatable and phosphomimetic versions of SAS-5 and tested their ability to interact with ZYG-1. We found that conversion of two residues to glutamate (phosphomimetic) blocked ZYG-1 binding, while conversion of all four residues to glutamate restored ZYG-1 binding. To confirm that this mechanism is important in vivo, we have used CRISPR-based gene editing to mutate the phosphorylated residues in the endogenous sas-5 gene. Surprisingly, none of the mutations completely block centriole assembly indicating that these phosphorylation events are not absolutely essential. However, a number of these mutations partially block centriole assembly and thus phosphorylation of these residues are likely to contribute to the efficiency of centriole assembly. Most recently we have taken a complimentary approach by using CRISPR to mutate conserved serine and threonine residues in SAS-5 to alanine and have found that serine 10 is essential for SAS-5 function. Further, we find that three serine residues (S331, 338, and 340) at the C. terminus of SAS-5 are also essential. Using multidimensional confocal imaging we have found that conversion of serine 10 to either alanine (non-phosphorylated mutant) or glutamate (phosphomimetic mutant) results in a sas-5 loss-of-function phenotype, whereby centriole duplication fails at a high rate. However, the S10A non-phosphorylatable mutant exhibits a near complete bock in centriole assembly while the S10E phosphomimetic mutant is frequently able to duplicate its centrioles. This suggests that the S10E mutation partially mimics a phosphorylated residue. Using quantitative immunoblotting, we have found that the S10A and the S10E forms of SAS-5 are expressed at normal levels, indicating that blocking or mimicking phosphorylation of this residue results in a nonfunctional SAS-5 protein rather than an unstable protein. Finally, we have also analyzed a triple mutant (S331A, S338A, and S340A) and found that it causes the production of excess centrioles. Quantitative western blots indicate that this SAS-5-A mutant is overexpressed two-fold relative to wild-type SAS-5. Interestingly, both the centriole overduplication defect and the over-expression defect can be rescued by co-expressing a wild-type version of SAS-5. This indicates that phosphorylation of these residues down-regulates expression of SAS-5 and that this mechanism can operate in trans to control unphosphorylated SAS-5 proteins. To understand how failure to phosphorylate serine 10 affects centriole assembly, we have performed a recruitment assay to follow the behavior of the SAS-5-SAS-6 complex in wild-type and SAS-5S10A embryos during the process of centriole assembly. We find that loss of phosphorylation of serine 10 does not affect the initial recruitment of the SAS-5-SAS-6 complex. However, whereas wild-type SAS-5-SAS-6 is stably incorporated into growing centrioles, loss of serine 10 phosphorylation results in dispersal of SAS-5-SAS-6. This is similar to the effect of blocking ZYG-1 kinase activity, suggesting that phosphorylation of serine 10 by ZYG-1 is a critical event in centriole assembly. As mentioned above we are also interested in the mechanisms underlying centriole stability. SAS-1 is a C2 domain containing centriole component that plays an important role in maintaining the structural integrity of centrioles. In its absence centrioles can form but ultimately disassemble. How SAS-1 stabilizes centrioles and how its activity might be regulated to control cell-type specific centriole elimination is unknow. To begin to address these questions, we have taken a genetic approach. The sas-1(t1476) mutation is a temperature-sensitive mutation that confers a fully penetrant embryonic lethal phenotype at the restrictive temperature. Using a genetic screening approach similar to that used to identify zyg-1 suppressors, we have identified a number of mutations that restore embryonic viability to the sas-1(1476) mutant. Most of these are intragenic (the mutations are in sas-1 itself) and somehow restore SAS-1 function. However five of the suppressors appear to be extragenic and likely identify genes with previously unknown roles in centriole stability. We are currently applying genomic sequencing and a positional mapping strategy to identify the mutated genes.