Figure 3.
PLK-1 phosphorylation controls a direct interaction between SPD-5 and the γ-tubulin complex.(A) Reconstitution of the C. elegans γ-tubulin complex (schematic) by coexpression in mammalian cells (flowchart in middle). Plasmids encoding γ-tubulin, GIP-1, GIP-2, and MZT-1 were cotransfected, and the Myc tag on GIP-2 was used to immunoprecipitate the complex (Myc IP). Immunoblots show input (left blot, α-tubulin [α-tub] was used as a loading control) and anti-Myc immunoprecipitated material (right blot) for the indicated conditions. (B) Schematic showing the locations in SPD-5 of the clusters of PLK-1 phosphorylation sites (indicated by red circled P) implicated in γ-tubulin recruitment (Cluster II; S170, T178, and T198) and PCM matrix expansion (S653 and S658). A SPD-5N fragment that lacks the PCM expansion sites was bacterially expressed and purified. Blot shows effect of incubation of SPD-5N variants with or without PLK-1 in the presence of ATP for 1 h at 23°C. (C) Left: Schematic of analysis of interaction of γ-tubulin complexes immobilized on beads, with SPD-5N (either WT or Cluster IImut) preincubated with or without PLK-1. Right: Blots of bead-bound components after incubation and isolation are shown; FLAG-tagged MZT-1 was present but was not blotted here. (D) Analysis of interaction of PLK-1–phosphorylated SPD-5N with γ-tubulin complexes reconstituted with or without FLAG-tagged MZT-1. (E) Graph of embryonic lethality (mean ± SD); N refers to number of worms and n to number of embryos scored. (F) Top: Alignment of sequences from divergent nematodes highlighting potential conservation of the critical T198 site. Bottom: Indicated SPD-5N variants incubated with or without PLK-1 and immunoblotted to detect total SPD-5 (top) and phosphorylation of T198 (bottom), using a phospho-specific antibody (pT198). (G) Analysis of γ-tubulin complex binding, conducted as in C, for the indicated conditions. (H and I) Representative images and quantification of γ-tubulin::mCherry (mCh) recruitment to centrosomes for the indicated conditions. The red asterisk marks the location of the T198A mutation in SPD-5. Dashed line marks the amount of γ-tubulin at centrosomes at nuclear envelope breakdown (NEBD) in embryos expressing WT SPD-5. Error bars are the 95% confidence interval. Times are in seconds after NEBD. Scale bar, 2 µm. Data for WT and Cluster IImut SPD-5 is reproduced from Fig. 2, E and F for comparison. (J) Embryonic lethality is plotted for the indicated conditions. Data for the non–gfp-containing transgene (None, WT, and Expmut) conditions is the same as that shown in Fig. 1 D and is reproduced here for comparison. N refers to the number of worms and n to the number of embryos scored. Error bars are the standard deviation. B. malayi, Brugia malayi; C. brenneri, Caenorhabditis brenneri; O. volvulus, Onchocerca volvulus.

PLK-1 phosphorylation controls a direct interaction between SPD-5 and the γ-tubulin complex. (A) Reconstitution of the C. elegans γ-tubulin complex (schematic) by coexpression in mammalian cells (flowchart in middle). Plasmids encoding γ-tubulin, GIP-1, GIP-2, and MZT-1 were cotransfected, and the Myc tag on GIP-2 was used to immunoprecipitate the complex (Myc IP). Immunoblots show input (left blot, α-tubulin [α-tub] was used as a loading control) and anti-Myc immunoprecipitated material (right blot) for the indicated conditions. (B) Schematic showing the locations in SPD-5 of the clusters of PLK-1 phosphorylation sites (indicated by red circled P) implicated in γ-tubulin recruitment (Cluster II; S170, T178, and T198) and PCM matrix expansion (S653 and S658). A SPD-5N fragment that lacks the PCM expansion sites was bacterially expressed and purified. Blot shows effect of incubation of SPD-5N variants with or without PLK-1 in the presence of ATP for 1 h at 23°C. (C) Left: Schematic of analysis of interaction of γ-tubulin complexes immobilized on beads, with SPD-5N (either WT or Cluster IImut) preincubated with or without PLK-1. Right: Blots of bead-bound components after incubation and isolation are shown; FLAG-tagged MZT-1 was present but was not blotted here. (D) Analysis of interaction of PLK-1–phosphorylated SPD-5N with γ-tubulin complexes reconstituted with or without FLAG-tagged MZT-1. (E) Graph of embryonic lethality (mean ± SD); N refers to number of worms and n to number of embryos scored. (F) Top: Alignment of sequences from divergent nematodes highlighting potential conservation of the critical T198 site. Bottom: Indicated SPD-5N variants incubated with or without PLK-1 and immunoblotted to detect total SPD-5 (top) and phosphorylation of T198 (bottom), using a phospho-specific antibody (pT198). (G) Analysis of γ-tubulin complex binding, conducted as in C, for the indicated conditions. (H and I) Representative images and quantification of γ-tubulin::mCherry (mCh) recruitment to centrosomes for the indicated conditions. The red asterisk marks the location of the T198A mutation in SPD-5. Dashed line marks the amount of γ-tubulin at centrosomes at nuclear envelope breakdown (NEBD) in embryos expressing WT SPD-5. Error bars are the 95% confidence interval. Times are in seconds after NEBD. Scale bar, 2 µm. Data for WT and Cluster IImut SPD-5 is reproduced from Fig. 2, E and F for comparison. (J) Embryonic lethality is plotted for the indicated conditions. Data for the non–gfp-containing transgene (None, WT, and Expmut) conditions is the same as that shown in Fig. 1 D and is reproduced here for comparison. N refers to the number of worms and n to the number of embryos scored. Error bars are the standard deviation. B. malayi, Brugia malayi; C. brenneri, Caenorhabditis brenneri; O. volvulus, Onchocerca volvulus.

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