Panel A: A bar graph showing the number of centriole pairs for different genotypes of Ana1. The x-axis represents different Ana1 genotypes, and the y-axis represents the number of centriole pairs. The graph indicates that there is no significant difference in the number of centriole pairs among the different genotypes. Panel B: A Western blot analysis showing the interaction between Ana1 and Plk1. The blot includes lanes for different combinations of Ana1 and Plk1, with labels for MBP-Ana1 (IP), GST-PBD (IP), and MBP-Ana1 (Input). Panel C: A series of microscopy images showing centriole elongation in primary spermatocytes at various stages. The images are stained for EGFP-Ana1 (green) and Asl (red), with different genotypes of Ana1. The stages include Duplicated, Early-Elongation, Mid-Elongation, and Mature Primary Spermatocyte. Panel D: A Coomassie-stained SDS-PAGE gel showing the binding of His-Ana1 fragments to tubulin. The gel includes lanes for His-Ana1 1-497 and His-Ana1 139-497, with and without tubulin. Panel E: Microscopy images showing the binding of tubulin to different fragments of Ana1 and Cep135. The images are labeled with different combinations of tubulin, Cep135, and Ana1 fragments.
MT binding–deficient Ana1 mutants fail to induce centriole overelongation. (A) Representative images showing centriole elongation in primary spermatocytes at various stages from different genotypes, stained for EGFP-Ana1 (green) and Asl (red). Scale bar, 2 µm. (B) Centriole pairs, marked by Asl localization, were quantified in 15–20 cells per testis from the indicated genotypes. Each data point represents the number of centriole pairs per testis, derived from five independent testes per genotype. Error bars indicate the mean ± SD. ns, not significant (unpaired two-tailed Student’s t test). (C)In vitro GST pull-down assay showing interactions between MBP-Ana11404–1729 or Ana11404–1729RK and GST-PBD when incubated with or without PLK (indicated as “Plk1 +/−”). Top panel, western blot analysis of MBP-Ana1 being pulled down by GST-PBD. Note that the binding was enhanced by PLK1 phosphorylation and the RK mutation had no effect on protein binding. Middle panel, SDS-PAGE analysis showing comparable amount of GST-PBD coupled to the GST beads. Bottom panel, SDS-PAGE analysis showing MBP-Ana1 input. Asterisks highlight the degradation band of MBP-Ana1. (D) Coomassie blue–stained SDS-PAGE gel of a MT cosedimentation experiment performed with 2 mg/ml polymerized tubulins and His-Ana11–497 or His-Ana1139–497(CR1 deletion). (E) Rhodamine-labeled tubulin (red; ∼10% labeling ratio with unlabeled tubulin) was mixed with 1 μM Taxol-stabilized MTs in combination with 1 μM Cep1351–498, 1 μM Ana1180–497, 1 μM Ana1Δ282–301, or 1 μM Ana1RKN as indicated. Identical imaging settings were used across all groups for direct comparison. Scale bar, 20 μm. PBD, Polo-box domain. Source data are available for this figure: SourceData FS5.