Protein phosphatase 2A-SUR-6/B55 regulates centriole duplication in C. elegans by controlling the levels of centriole assembly factors

Abstract

Centrioles play a crucial role in mitotic spindle assembly and duplicate precisely once per cell cycle. In worms, flies, and humans, centriole assembly is dependent upon a key regulatory kinase (ZYG-1/Sak/Plk4) and its downstream effectors SAS-5 and SAS-6. Here we report a role for protein phosphatase 2A (PP2A) in centriole duplication. We find that the PP2A catalytic subunit LET-92, the scaffolding subunit PAA-1, and the B55 regulatory subunit SUR-6 function together to positively regulate centriole assembly. In PP2A-SUR-6-depleted embryos, the levels of ZYG-1 and SAS-5 are reduced and the ZYG-1- and SAS-5-dependent recruitment of SAS-6 to the nascent centriole fails. We show that PP2A physically associates with SAS-5 in vivo and that inhibiting proteolysis can rescue SAS-5 levels and the centriole duplication defect of PP2A-depleted embryos. Together, our findings indicate that PP2A-SUR-6 promotes centriole assembly by protecting ZYG-1 and SAS-5 from degradation.

Figure 1. PP2Ac^LET-92 -SUR-6 regulates centriole duplication

(A) PP2Ac^LET-92 coprecipitates with SZY-20. (B) Depletion of PP2Ac^LET-92 results in a decrease in SZY-20 mobility consistent with hyperphosphorylation. (C) PP2Ac^LET-92 coprecipitates with SUR-6. (D–I) Embryos of indicated genotype stained for microtubules (red), centrioles (SAS-4, green), and DNA (blue). Depletion of SUR-6 in wild-type embryos (G) results in centriole duplication defects such as monopolar spindles (left blastomere) or asymmetric spindles (right blastomere). Insets show symmetrical staining of wild-type spindle poles (D) and asymmetrical staining of sur-6(RNAi) spindle poles (G). (H, I) RNAi of sur-6 enhances the centriole duplication defect of zyg-1(it25) embryos raised at permissive temperature. (J) A SUR-6 immunoblot demonstrating the specificity of the α-SUR-6 antibody. RNAi of sur-6 results in a reduction in the intensity of the SUR-6 band. (K, L) Immunostaining reveals a broad distribution of SUR-6 (green) in the control embryo (K) and a loss of this staining in the sur-6(RNAi) embryo (L). Lower panels show the same embryos stained for microtubules (red) and DNA (blue). Bars, 10μm. For supporting data see Figure S1, Table S1, and movies S1 and S2.

Figure 2. PP2Ac^LET-92 genetically interacts with other centriole duplication factors

(A) A genetic interaction network of centriole duplication genes. Shown is the percent embryonic lethality of indicated double heterozygotes. Single heterozygotes are listed in last column (+). Some single homozygotes exhibit a sterile uncoordinated (Stu) phenotype and others a larval lethal (Lva) phenotype. Between 345 and 1,521 embryos were counted for each strain. (B–E) Embryos of indicated genotype stained for microtubules (red), centrioles (SAS-4, green), and DNA (blue). Note that let-92 (B) and zyg-1 heterozygotes (not shown) assemble bipolar spindles, but zyg-1 homozygotes (C), and let-92/+; zyg-1/+ (D) and let-92/+; +/sas-5 double heterozygotes assemble monopolar spindles. Bar, 10 μm. For supporting data see Movie S3.

Figure 3. PP2Ac^LET-92 is required for recruitment of the SAS-5/6 complex

(A) Control and (B) let-92(RNAi) embryos stained for ZYG-1 (red), GFP-SAS-4 (green) and DNA (blue). ZYG-1 localizes to centrosomes in PP2Ac^LET-92 -depleted embryos (insets). (C) Control and (D) let-92(RNAi) embryos stained for endogenous SAS-4 (red) GFP-SAS-6 (green-insets only) and DNA (blue). (C) In control embryos, GFP-SAS-6 is recruited to centrioles as evidence by the overlap between the GFP-SAS-6 and SAS-4 signals (insets). (D) In let-92(RNAi) embryos, GFP-SAS-6 does not accumulate around the centrioles. Note that control and let-92(RNAi) embryos are approximately the same age based on position of DNA and separation of centrioles. The DNA of let-92(RNAi) embryos however stays highly condensed.

Figure 4. The PP2Ac^LET-92 -SUR-6 complex regulates the levels of ZYG-1 and SAS-5

(A–C) Quantitative immunoblots of embryonic extracts showing that RNAi of let-92, sur-6, or paa-1 reduces the levels of ZYG1 and SAS-5 but not the levels of SAS-6. (D) PP2Ac^LET-92 coprecipitates with SAS-5, but not with SAS-6. (E) Weak rpt-4(RNAi) restores SAS-5 levels in PP2Ac^LET-92-depleted embryos. Shown is a graph depicting the average relative levels of SAS-5 with standard deviations from three experiments, and a representative blot. The reduction of SAS-5 levels upon let-92(RNAi) is less pronounced in these experiments due to dilution of let-92 bacteria with control bacteria. Ratios of RNAi bacteria used: let-92:control, 1:1; let-92:rpt-4:control: 4:1:3; rpt-4:control, 1:7. For supporting data see movie S4.