Figure 4.
Inhibiting γ-tubulin docking and PCM expansion by mutating substrate target sites recapitulate the effects of loss of centrosomal PLK-1 on the ability of centrosomes to catalyze spindle assembly. (A and B) Images of GFP::β-tubulin (green) and mCherry::histone (magenta) in embryos expressing the indicated untagged SPD-2 (A) or SPD-5 (B) variants with endogenous SPD-2 (A) or SPD-5 (B) depleted. The protein sequence between amino acids 232 and 234 was changed from STP to TAP in PDmut SPD-2 as indicated. Red asterisks mark the mutated phosphorylation site clusters in SPD-5. Times in the top left of each panel are seconds after NEBD. Percentages of chromosome segregation errors are in yellow at the bottom right of the last panel of each sequence. Chromosome segregation data for conditions other than the double mutant are the same as that shown in Fig. 1 D and Fig. 2 J and are shown here for comparison. Scale bars are 5 µm. (C) Quantification of centrosome separation (left) or centrosomal β-tubulin intensity (right) for the indicated conditions. Error bars are the 95% confidence intervals. N refers to number of embryos and n to number of centrosomes scored. (D) Schematic depicting independent phospho-regulation by centrosome-localized PLK-1 of γ-tubulin recruitment and PCM expansion through phosphorylation of PLK-1 site clusters (red circled P) in distinct SPD-5 regions. Black circles and other symbols in the schematics are defined as in Fig. 1 A. (E) Sequence alignments supporting nematode SPD-5s being divergent orthologues of CDK5RAP2 and Cnn. SPD-5s from distantly related nematodes (∼400 Mya apart) have a CM1-like motif N-terminal to the key phospho-regulated sites identified here (see Fig. 3 F). SPD-5 also has a CM2-like motif at its C-terminus (Fig. S3 C) and a central PReM-like region (Fig. S3 D). (F) Speculative model for mechanism of phospho-regulated γ-tubulin complex recruitment. The CM1-like recruitment site is masked in unphosphorylated cytoplasmic SPD-5. Phosphorylation at centrosomes unmasks the binding site, leading to γ-tubulin complex recruitment specifically on PCM matrix-assembled SPD-5. B. malayi, Brugia malayi; G. gallus, Gallus gallus; H. sapiens, Homo sapiens; PReM, phospho-regulated multimerization. NEBD, nuclear envelope breakdown; Bkgd, background; ASH; ASPM; SPD-2, Hydin domain.

Inhibiting γ-tubulin docking and PCM expansion by mutating substrate target sites recapitulate the effects of loss of centrosomal PLK-1 on the ability of centrosomes to catalyze spindle assembly. (A and B) Images of GFP::β-tubulin (green) and mCherry::histone (magenta) in embryos expressing the indicated untagged SPD-2 (A) or SPD-5 (B) variants with endogenous SPD-2 (A) or SPD-5 (B) depleted. The protein sequence between amino acids 232 and 234 was changed from STP to TAP in PDmut SPD-2 as indicated. Red asterisks mark the mutated phosphorylation site clusters in SPD-5. Times in the top left of each panel are seconds after NEBD. Percentages of chromosome segregation errors are in yellow at the bottom right of the last panel of each sequence. Chromosome segregation data for conditions other than the double mutant are the same as that shown in Fig. 1 D and Fig. 2 J and are shown here for comparison. Scale bars are 5 µm. (C) Quantification of centrosome separation (left) or centrosomal β-tubulin intensity (right) for the indicated conditions. Error bars are the 95% confidence intervals. N refers to number of embryos and n to number of centrosomes scored. (D) Schematic depicting independent phospho-regulation by centrosome-localized PLK-1 of γ-tubulin recruitment and PCM expansion through phosphorylation of PLK-1 site clusters (red circled P) in distinct SPD-5 regions. Black circles and other symbols in the schematics are defined as in Fig. 1 A. (E) Sequence alignments supporting nematode SPD-5s being divergent orthologues of CDK5RAP2 and Cnn. SPD-5s from distantly related nematodes (∼400 Mya apart) have a CM1-like motif N-terminal to the key phospho-regulated sites identified here (see Fig. 3 F). SPD-5 also has a CM2-like motif at its C-terminus (Fig. S3 C) and a central PReM-like region (Fig. S3 D). (F) Speculative model for mechanism of phospho-regulated γ-tubulin complex recruitment. The CM1-like recruitment site is masked in unphosphorylated cytoplasmic SPD-5. Phosphorylation at centrosomes unmasks the binding site, leading to γ-tubulin complex recruitment specifically on PCM matrix-assembled SPD-5. B. malayi, Brugia malayi; G. gallus, Gallus gallus; H. sapiens, Homo sapiens; PReM, phospho-regulated multimerization. NEBD, nuclear envelope breakdown; Bkgd, background; ASH; ASPM; SPD-2, Hydin domain.

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