Figure S3.
Related to Fig. 4: In situ–tagged GIP-2::GFP exhibits a synthetic spindle assembly phenotype when combined with Cluster IImut SPD-5. (A) Example images of GIP-2::GFP in the presence of WT or Cluster IImut SPD-5. Times in the top left of large panels and below smaller panels are seconds relative to NEBD. Scale bar, 2 µm. Error bars are mean with 95% confidence intervals. P values are from two-tailed t tests. Red asterisk marks the location of the Cluster II mutation in SPD-5. (B) Quantification of centrosome separation for the indicated conditions. Spindle length was measured using mCherry (mCh)::SAS-4 that was also expressed in the analyzed strains. Times are seconds after NEBD. Error bars are the 95% confidence intervals. N refers to number of embryos imaged. Red asterisk marks the location of the Cluster II mutation in SPD-5. Scale bar, 3 μm. (C and D) Nematode SPD-5s have regions with homology to the domains of Drosophila Cnn that mediate its PLK-1–regulated self-assembly. Expansion of the Cnn-based PCM matrix occurs through a phospho-regulated self-interaction in which a conserved CM2 motif at the Cnn C-terminus interacts with an internal region of Cnn (called the phospho-regulated multimerization or “PReM” domain) composed of a leucine zipper followed by a control region that enables assembly when phosphorylated by PLK-1 (Citron et al., 2018; Conduit et al., 2014; Feng et al., 2017). (C) Sequence alignment suggesting the presence of a CM2-like domain at the SPD-5 C-terminus. (D) Sequence alignment showing the region of SPD-5 containing the PLK-1 sites previously shown to control SPD-5 self-assembly (S653 and S658; Woodruff et al., 2015). Pink asterisks mark candidate PLK-1 sites predicted by GPS (Group-based Prediction System) Polo (Xue et al., 2005) with a score >2.165. In preliminary work (not shown), we found that mutant VI (Fig. 2A and Fig. S1 E) also leads to an Expmut-like phenotype, suggesting that some of the other PLK-1 sites in this region may also be essential for PCM assembly. The locations of the PLK1 site clusters that control the assembly of C. elegans SPD-5 and Drosophila Cnn are indicated on the schematics with red-circled Ps. Like the PLK-1 sites in the PReM region required for Cnn assembly (Conduit et al., 2014), the functionally important PLK1 sites in the C. elegans protein follow a short leucine-rich region. NEBD, nuclear envelope breakdown; PReM, phospho-regulated multimerization. B. malayi, Brugia malayi; G. gallus, Gallus gallus; H. sapiens, Homo sapiens.

Related to Fig. 4: In situ–tagged GIP-2::GFP exhibits a synthetic spindle assembly phenotype when combined with Cluster IImut SPD-5. (A) Example images of GIP-2::GFP in the presence of WT or Cluster IImut SPD-5. Times in the top left of large panels and below smaller panels are seconds relative to NEBD. Scale bar, 2 µm. Error bars are mean with 95% confidence intervals. P values are from two-tailed t tests. Red asterisk marks the location of the Cluster II mutation in SPD-5. (B) Quantification of centrosome separation for the indicated conditions. Spindle length was measured using mCherry (mCh)::SAS-4 that was also expressed in the analyzed strains. Times are seconds after NEBD. Error bars are the 95% confidence intervals. N refers to number of embryos imaged. Red asterisk marks the location of the Cluster II mutation in SPD-5. Scale bar, 3 μm. (C and D) Nematode SPD-5s have regions with homology to the domains of Drosophila Cnn that mediate its PLK-1–regulated self-assembly. Expansion of the Cnn-based PCM matrix occurs through a phospho-regulated self-interaction in which a conserved CM2 motif at the Cnn C-terminus interacts with an internal region of Cnn (called the phospho-regulated multimerization or “PReM” domain) composed of a leucine zipper followed by a control region that enables assembly when phosphorylated by PLK-1 (Citron et al., 2018; Conduit et al., 2014; Feng et al., 2017). (C) Sequence alignment suggesting the presence of a CM2-like domain at the SPD-5 C-terminus. (D) Sequence alignment showing the region of SPD-5 containing the PLK-1 sites previously shown to control SPD-5 self-assembly (S653 and S658; Woodruff et al., 2015). Pink asterisks mark candidate PLK-1 sites predicted by GPS (Group-based Prediction System) Polo (Xue et al., 2005) with a score >2.165. In preliminary work (not shown), we found that mutant VI (Fig. 2A and Fig. S1 E) also leads to an Expmut-like phenotype, suggesting that some of the other PLK-1 sites in this region may also be essential for PCM assembly. The locations of the PLK1 site clusters that control the assembly of C. elegans SPD-5 and Drosophila Cnn are indicated on the schematics with red-circled Ps. Like the PLK-1 sites in the PReM region required for Cnn assembly (Conduit et al., 2014), the functionally important PLK1 sites in the C. elegans protein follow a short leucine-rich region. NEBD, nuclear envelope breakdown; PReM, phospho-regulated multimerization. B. malayi, Brugia malayi; G. gallus, Gallus gallus; H. sapiens, Homo sapiens.

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