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 number. More recently as part of this project we have focus on the genetic elements 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 at least partially restore embryonic viability to the sas-1(1476) mutant. Genomic sequencing revealed that all such suppressor mutation are intragenic (i.e. in the sas-1 gene) and thus likely act by directly restoring SAS-1 function. Furthermore all suppressing mutations map to the same C2 domain as the original sas-1(t1476) mutation. This past year we have carefully analyzed these intragenic suppressors and have found that they restore centriole stability to the original sas-1(t1476) mutant. More specifically these second site sas-1 mutations partially or completely restore the ability to assemble bipolar spindles and suppress the tendency of sas-1(t1476) mutant centrioles to fragment. One of these suppressors restores viability and bipolar spindle assembly to wild-type levels while the other three mutations provide partial suppression. As part of this project we are also analyzing the role of a second C2 domain which is located near the N-terminus of SAS-1. We find that a clean deletion of this domain results in a strong loss-of-function phenotype and that the resulting protein can still weakly localize to centrioles. We are continuing to analyze this mutant to define the role of this C2 domain in maintaining centriole stability. After assessing the results of our initial sas-1 suppressor screen, we came to the conclusion that the original allele was suboptimal for identifying extragenic mutations. We therefore repeated the screen, this time using one of the sas-1 double mutants identified in the initial screen. The sas-1(t1476 bs272) mutant, while able to grow better than the sas-1(t1476) mutant, still can’t grow at 25.5°C. Using our standard screening strategy we identified 32 new suppressor mutations that allow growth of the sas-1(t1476 bs272) mutant at 25.5°. Interestingly only 3 are these suppressor mutations are intragenic. Of the remaining suppressors, three carry a mutation in a centriole gene (one each in sas-6, sas-5, and sas-4). Several suppressors also carry mutations in PCM components (spd-5 and pcmd-1), suggesting that altering PCM activity can restore centriole stability to the sas-1 mutant. In addition, 15 suppressors carry mutations in the same novel gene, which has no known connection with the centriole. Using a CRISPR-based genome editing approach we have confirmed that the mutations in sas-4, sas-6, spd-5 and pcmd-1 are all bona fide suppressors of the sas-1(t1476 bs272) embryonic lethal phenotype. Conversely, we have found that the mutation in sas-5 is not a suppressor. Additionally we confirmed that the novel gene hit multiple times in our screen is also a suppressor. This gene which we will refer to as sup-1 encodes an adapter for an E3 ubiquitin ligase. Interestingly one of the other suppressor lines carries a mutation in an E3 ubiquitin ligase that is known to physically interact with the product of the sup-1 gene. Using our CRISPR-based approach we have confirmed that the mutation in the ubiquitin ligase is also a suppressor. As we have localized both the SUP-1 protein and the ubiquitin ligase to centrosomes, this E3 ubiquitin ligase complex may be directly involved in targeting centrosome proteins for destruction. To identify the relevant substrates of this ubiquitin ligase we have taken a candidate-based approach and have analyzed centrosome-associated factors. So far we find no evidence that this ubiquitin ligase targets SAS-1, SPD-5, or SPD-2. We plan to continue with our current strategy of analyzing centrosome proteins while also taking an unbiased proximity labeling approach.