Transgene replacement systems for SPD-2 and SPD-5, method for identifying putative PLK-1 sites in SPD-5, and analysis of PLK-1 and SPD-2 centrosomal localization in Cluster II^mut and Exp^mut SPD-5. (A and B) Single-copy RNAi-resistant transgene insertion systems used to express SPD-2 (A) and SPD-5 (B) variants. Regions marked “RR” were altered in nucleotide but not in protein coding sequence to make the transgene-encoded products resistant to dsRNAs targeting the equivalent region of the endogenous genes. Due to the repetitive architecture of the spd-5 promoter region, the spd-2 promoter was used to control expression of the spd-2 and spd-5 transgenes. (C) Protocol employed to assess embryonic lethality. L4 worms were injected with dsRNA targeting spd-5 or spd-2, and embryos laid 48–72 h (at 16°C) after injection were scored for lethality. (D) Outline of procedure used to identify putative PLK-1 target sites in SPD-5. Red asterisk marks the phosphorylation site. (E) Schematic depicting identified sites and regional clusters employed for mutagenesis. Seven transgenes harboring mutant clusters I–VII were used to generate single-copy integration strains that were used for the embryonic lethality analysis shown in Fig. 2 A. (F) Images of in situ–tagged mCherry::SPD-2 (top) and PLK-1::GFP (bottom) in embryos expressing WT or Cluster II^mut SPD-5 after endogenous SPD-5 depletion. Red asterisks mark the Cluster II mutation site in SPD-5. Times in the top left of large panels and below smaller panels are in seconds relative to NEBD. Scale bars, 2 µm. Graphs on right show quantification of centrosomal mCherry (mCh)::SPD-2 or PLK::GFP signal at NEBD. Error bars are mean with 95% confidence intervals. P values are from two-tailed t tests. (G) Centrosomal fluorescence of mCherry::SPD-2 (top) and PLK-1::GFP (bottom) in embryos expressing WT or Exp^mut SPD-5 after endogenous SPD-5 depletion. Values were plotted after normalizing to the mean at NEBD for WT SPD-5. Error bars are the SEM; n is the number of centrosomes quantified. SPD-2 is recruited to centrioles by binding to the C-terminus of SAS-7 (Sugioka et al., 2017) and is thought to associate with the PCM matrix via a direct association with SPD-5 (Boxem et al., 2008). Consistent with this, levels of centrosomal mCherry::SPD-2 and PLK-1::GFP are similar in embryos expressing WT and Exp^mut SPD-5 before mitotic entry and expansion of the SPD-5 matrix. After mitotic entry, mCherry::SPD-2 levels at centrosomes increase as the WT SPD-5 matrix expands, and this increase fails to occur in Exp^mut SPD-5 embryos. unc, uncoordinated; NEBD, nuclear envelope breakdown.