Panel A: A western blot image showing protein bands for different genotypes. The blot is probed with anti-Ana1 and anti-tubulin antibodies. Panel B: Violin plots showing the number of pupae produced by different genotypes, with statistical significance indicated. Panel C: Microscopy images of centrioles stained for EGFP-Ana1, Asl, and DNA, with insets showing detailed views. Panel D: Microscopy images of centrioles stained for EGFP-Ana1, Cep135-mRuby3, and DNA, with insets showing detailed views. Panel E: Bar graphs showing the mean intensity of Ana1 and Cep135 per unit length of centrioles, with error bars indicating standard deviation. Panel F: Dot plots showing the lengths of centrioles marked by Ana1, Asl, or Cep135, with error bars indicating standard deviation and statistical significance noted. Panel G: Transmission electron microscopy images showing cross-sections and longitudinal sections of centrioles in Drosophila primary spermatocytes, highlighting ninefold symmetry and centriole shortening.
Ana1-MT interaction is essential for Ana1-mediated centriole elongation in primary spermatocytes. (A) Western blot of extracts from 50 to 60 testes for indicated genotypes. Immunoblot analysis of Ana1 protein variants with an anti-Ana1 primary antibody. α-Tubulin is shown as a loading control. (B) Graphs showing the male fertility of different genotypes, as measured by the number of progeny pupae produced from mating with WT (w1118) virgin females. ****P < 0.0001. ns, not significant. (C and D) Centrioles in primary spermatocytes stained to reveal EGFP-Ana1 (green), Asl (red), Cep135-mRuby3 (red), and DNA (blue). Scale bar, 10 µm (overview image) or 2 µm (inset image). (E) Graphs showing the mean ± SD quantification for Ana1 and Cep135 mean intensity per unit length of centrioles in primary spermatocytes. Each point on the graph represents the average mean intensity measured from a 16-cell cyst with 10–15 centrioles scored; five independent testes were measured for each genotype with 2–3 cell cysts scored in each testis. ns, not significant. (F) Graphs showing the mean ± SD quantification for centriole lengths in mature primary spermatocytes using Ana1, Asl, or Cep135 as a centriole marker for each genotype. Each point on the graphs represents the average length measured from a 16-cell cyst with 10–15 centrioles scored; five independent testes were measured for each genotype with 2–3 cell cysts scored in each testis. *P < 0.05, **P < 0.01, ****P < 0.0001. ns, not significant. (G) Cross-sections (left) of centrioles in Drosophila primary spermatocytes by TEM showing preserved ninefold symmetry in both EGFP-Ana1 (top) and EGFP-Ana1-RKNC (bottom) groups. Longitudinal sections (right) show that the RKNC mutation leads to centriole shortening compared with full-length Ana1 controls. For each genotype, centrioles were examined from at least three independent testes, with a total of 8–12 centrioles analyzed. Scale bar, 200 nm. WT, wild type. Source data are available for this figure: SourceData F8.