The extreme N-terminal region of Cnn-C inhibits binding to γ-tubulin complexes. (A) Diagram of the Cnn-C and testes-specific Cnn (Cnn-T) isoforms that exist in vivo. (B) Diagram of artificial Cnn proteins with differing N-terminal regions used to form Cnn scaffolds (induced by phosphomimetic mutations in the PReM domain; beige) via mRNA injection into unfertilized eggs. (C–F) Fluorescence images of unfertilized eggs expressing γ-tubulin37C-mCherry that were injected with mRNA encoding different types of artificial Cnn proteins, as indicated. Insets show representative examples of individual scaffolds. (G) Graph showing fluorescence intensity measurements (in arbitrary units) of γ-tubulin37C-mCherry and GFP-Cnn at Cnn-C (n = 1,498 scaffolds; 12 eggs), Cnn-T (n = 1,400 scaffolds; 10 eggs), Cnn-C^Δ1-77 (n = 2,168 scaffolds; 10 eggs), or Cnn-C^Δ1-97 (n = 400 scaffolds; 7 eggs) scaffolds. Each dot represents a single scaffold. (H) Graph shows slope values of linear regression lines calculated for scaffolds of different types. Each slope value represents an individual egg that contained multiple scaffolds. The geometric mean and 95% CIs are indicated. P values are from comparisons to the Cnn-C mean using a one-way ANOVA of log₁₀ transformed data. (IandJ) Western blot of a co-IP experiment (I) and quantification of γ-tubulin bands (J) showing the efficiency with which different MBP-tagged N-terminal fragments of Cnn, as indicated, coimmunoprecipitate γ-tubulin from embryo extracts. γ-Tubulin band intensities were normalized within each of three experimental repeats to the γ-tubulin band in the respective MBP-Cnn-T-N IP.