Panel A shows a Western blot with bands representing Ana1 protein variants and a loading control. Panel B presents a violin plot depicting the number of progeny pupae produced by different genotypes, with axes labeled as Number of pupae and Genotype. Panel C includes immunofluorescence images of centrioles stained for EGFP-Ana1, Asl, and DNA, with scale bars indicating 10 micrometers for overview images and 2 micrometers for insets. Panel D shows similar immunofluorescence images but includes Cep135-mRuby3 staining. Panel E features dot plots quantifying centriole lengths using Ana1, Asl, or Cep135 as markers, with axes labeled as Length (micrometers) and Genotype. Panel F displays bar graphs quantifying the mean fluorescence intensity of Ana1 or Cep135 per unit length of centriole, with axes labeled as Intensity and Genotype. Panel G presents transmission electron microscopy images of centrioles in cross-sections and longitudinal sections, with scale bars indicating 200 nanometers.
Ana1-Cep135 interaction is essential for centriole length control in primary spermatocytes. (A) Western blot of extracts from 50 to 60 testes of 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 < 0.01, ***P < 0.001. (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 (insets). (E) 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 graph 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 < 0.01, ***P < 0.001, ****P < 0.0001. (F) Graphs showing the mean ± SD quantification of Ana1 or Cep135 mean fluorescence intensity (per unit length of centriole) for each genotype. 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. *P < 0.05. ns, not significant. (G) TEM of EGFP-Ana1 (top panels) and EGFP-Ana1Δ244–371 (bottom panels) in cross-sections (left) and longitudinal sections (right) of centrioles. Cross-sectional views reveal that the ninefold symmetric centriole architecture is preserved in both WT and mutant flies analyzed. Longitudinal sections show that full-length EGFP-Ana1 labels normal-length centrioles (top right), whereas deletion of the residues 244–371 results in shortened centrioles (bottom right). 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 F5.