Figure 4.
Phosphomimetic mutations within the CAI domain and downstream of the CM1 domain promote binding to γ-tubulin complexes.(A) A cartoon showing the N-terminal region (aa 1–255) of Cnn used in co-IP experiments. Regions of potential phosphorylation sites are indicated, with their amino acid sequence displayed. (B–F) Western blots of co-IP experiments (B,C, and E) and quantification of γ-tubulin bands (D and F) showing the efficiency with which different MBP-tagged N-terminal fragments of Cnn, as indicated, co-IP γ-tubulin from extracts of WT (B–F), or γ-tubulin (top panels in E) and Grip75GCP4-sfGFP (bottom panels in E) from extracts of Grip75GCP4-sfGFP–expressing embryos. In D and F, band intensities were normalized within each experiment to the γ-tubulin band in the respective MBP-Cnn-T-N IP. The connecting lines indicate data points obtained from within the same experiment. P values are from comparisons to the Cnn-C mean using either Wilcoxon matched-pairs signed rank tests (D; n = 9 for comparison with Cnn-C-NT27; n = 5 for comparison with Cnn-C-NP1) or a Dunn’s multiple comparisons test (F; n = 4). (G and H) Graphs showing the S values from eggs expressing either γ-tubulin-mCherry (G) or Grip75GCP4-sfGFP (H) which contain the indicated scaffold types. Note that the data for Cnn-C, Cnn-T, and Cnn-CΔ1-77 scaffolds is the same as in Fig. 1 H and Fig. 2 E to allow comparisons with the phosphomimetic scaffolds. In G: n = 2,650 scaffolds and 11 eggs for Cnn-CT27 scaffolds, 1,803 scaffolds and 11 eggs for Cnn-CT186 scaffolds, 2,482 scaffolds and 10 eggs for Cnn-CT173 scaffolds, and 2,835 scaffolds and 18 eggs for Cnn-CT27,S186 scaffolds. In H: n = 1,448 scaffolds and 10 eggs for Cnn-CT27 scaffolds, 1,074 scaffolds and 10 eggs for Cnn-CT186 scaffolds, and 943 scaffolds and 10 eggs for Cnn-CT27,S186 scaffolds. The geometric mean and 95% CIs are indicated. **, P < 0.01. P values were from comparisons to the Cnn-C mean using a one-way ANOVA of log10 transformed data. (I) Bar graph showing the results of a blind categorization of eggs containing the different scaffold types based on the ability of the scaffolds within each egg to organize microtubule asters (numbers of eggs analyzed indicated above). Note that the data for Cnn-C, Cnn-T, and Cnn-CΔ1-77 scaffolds is the same as in Fig. 3 D to allow comparisons with the phosphomimetic scaffolds.

Phosphomimetic mutations within the CAI domain and downstream of the CM1 domain promote binding to γ-tubulin complexes. (A) A cartoon showing the N-terminal region (aa 1–255) of Cnn used in co-IP experiments. Regions of potential phosphorylation sites are indicated, with their amino acid sequence displayed. (B–F) Western blots of co-IP experiments (B,C, and E) and quantification of γ-tubulin bands (D and F) showing the efficiency with which different MBP-tagged N-terminal fragments of Cnn, as indicated, co-IP γ-tubulin from extracts of WT (B–F), or γ-tubulin (top panels in E) and Grip75GCP4-sfGFP (bottom panels in E) from extracts of Grip75GCP4-sfGFP–expressing embryos. In D and F, band intensities were normalized within each experiment to the γ-tubulin band in the respective MBP-Cnn-T-N IP. The connecting lines indicate data points obtained from within the same experiment. P values are from comparisons to the Cnn-C mean using either Wilcoxon matched-pairs signed rank tests (D; n = 9 for comparison with Cnn-C-NT27; n = 5 for comparison with Cnn-C-NP1) or a Dunn’s multiple comparisons test (F; n = 4). (G and H) Graphs showing the S values from eggs expressing either γ-tubulin-mCherry (G) or Grip75GCP4-sfGFP (H) which contain the indicated scaffold types. Note that the data for Cnn-C, Cnn-T, and Cnn-CΔ1-77 scaffolds is the same as in Fig. 1 H and Fig. 2 E to allow comparisons with the phosphomimetic scaffolds. In G: n = 2,650 scaffolds and 11 eggs for Cnn-CT27 scaffolds, 1,803 scaffolds and 11 eggs for Cnn-CT186 scaffolds, 2,482 scaffolds and 10 eggs for Cnn-CT173 scaffolds, and 2,835 scaffolds and 18 eggs for Cnn-CT27,S186 scaffolds. In H: n = 1,448 scaffolds and 10 eggs for Cnn-CT27 scaffolds, 1,074 scaffolds and 10 eggs for Cnn-CT186 scaffolds, and 943 scaffolds and 10 eggs for Cnn-CT27,S186 scaffolds. The geometric mean and 95% CIs are indicated. **, P < 0.01. P values were from comparisons to the Cnn-C mean using a one-way ANOVA of log10 transformed data. (I) Bar graph showing the results of a blind categorization of eggs containing the different scaffold types based on the ability of the scaffolds within each egg to organize microtubule asters (numbers of eggs analyzed indicated above). Note that the data for Cnn-C, Cnn-T, and Cnn-CΔ1-77 scaffolds is the same as in Fig. 3 D to allow comparisons with the phosphomimetic scaffolds.

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