A novel function for CDK2 activity at meiotic crossover sites
Fig 2
Analysis of telomeric fusion defects in Cdk2T160A spermatocytes.
Chromosome spread preparations from adult (postnatal day 40) testes immunostained with ACA (green) and the telomeric shelterin protein RAP1 (red) in conjunction with SYCP3 (blue) are shown for WT (A–B), Cdk2T160A (C–D), and Cdk2−/− (E) for selected stages of meiotic prophase I. For each of the 3 genotypes, RAP1 and ACA can be detected at the telomeres and centromeres as specific foci, respectively. The centromeric ACA signal is always detected proximal to a telomeric RAP1 signal because of the proximity of the centrosome to telomeric ends in mouse chromosomes. In pachytene-like Cdk2−/− spermatocytes (E) and mid-pachytene–stage Cdk2T160A spermatocytes (D), telomere fusions can be observed. Quantification of fusion events (F) specifically for mid-pachytene–stage nuclei was performed for WT (orange bars, N = 3 total events counted) and Cdk2T160A spermatocytes (blue bars, N = 30 total events counted). Here, data are presented as the total number of NC–NC, NC–C, and C–C fusions, respectively. For each genotype, fusion events were counted from 90 images pooled from 3 biological replicates in which at least 20 images were taken from each replicate; no statistical test is applied because of the low numbers of countable events. Examples of C–C, NC–C, and NC–NC fusions are shown in panels D–E by pink arrows. Additional staining is shown for the pericentromeric chromatin marker, H3K9me3 (green) in conjunction with SYCP3 (blue) for WT (G–H), Cdk2T160A (I–J), and Cdk2−/− (K), for selected stages of meiotic prophase I. Histone H1t (red) positivity also shown and is indicative of mid-pachytene stage onwards. In all WT and Cdk2T160A images, pericentric chromatin can be visualized as a flare-like staining emanating from the end of bivalents. In Cdk2−/−, aggregates of H3K9me3-positive pericentromeric chromatin show the extensive interactions between centromeric ends. All images within Fig 2 are representative of at least 20 images taken for specified stages. Similar staining patterns were confirmed in at least 3 biological replicates. In all main panels, scale bars are representative of 5 μm; in all inset pictures, scale bars are representative of 1.25 μm. The underlying data for (F) can be found in S1 Data. ACA, autocentromere antibody; CDK2, cyclin-dependent kinase 2; C–C, centromeric end to centromeric end; H3K9me3, trimethylated lysine 9 of histone H3; NC–C, noncentromeric end to centromeric end; NC–NC, noncentromeric end to noncentromeric end; RAP1, TERF2 interacting protein; SYCP, synaptonemal complex protein; WT, wild-type.