A putative PLK-1 target site cluster in SPD-5 required to dock γ-tubulin complexes onto the PCM matrix. (A) Flowchart of screening strategy. Predicted PLK-1 target sites were mutated in seven clusters (schematic), and the consequences on embryonic lethality were assessed. Graph plots embryonic lethality (mean ± SD) for the indicated conditions; N refers to number of worms and n to number of embryos scored. (B–G) Images and quantification of centrosomal intensity of GFP::SPD-5 variants (B–D) or of γ-tubulin::mCherry (E–G) in the presence of the indicated untagged SPD-5 variants (red asterisks mark the locations of the Cluster II and Expansion blocking mutations); endogenous SPD-5 was depleted in all conditions. Centrosomal fluorescence was plotted after normalizing to the mean at nuclear envelope breakdown (NEBD) for WT GFP::SPD-5 (B–D) or for γ-tubulin::mCherry (mCh) in the presence of WT SPD-5 (E–G); the means for these controls at NEBD are marked with red dashed line in the graphs. Error bars are the 95% confidence intervals. n refers to number of centrosomes imaged for each condition. (H) Plot of the ratio of centrosomal γ-tubulin::mCherry to GFP::SPD-5 for the indicated mutants. Error bars are the 95% confidence intervals. P values are from two-tailed t tests; **, P < 0.01; ****, P < 0.0001. (I) Individual centrosomes from the time lapse sequences of embryos expressing WT and Cluster II^mut GFP::SPD-5 shown in B and C. Time points highlight the PCM expansion that occurs after anaphase onset when the WT GFP::SPD-5–based matrix disassembles under the influence of cortical pulling forces on matrix-anchored microtubules. (J) Chromosome segregation analysis for the indicated conditions. n refers to the number of embryos imaged. None, WT, and Exp^mut data are reproduced from Fig. 1 D for comparison. Scale bars, 2 µm.