Drosophila BubR1 is essential for meiotic sister-chromatid cohesion and maintenance of synaptonemal complex

Abstract

The partially conserved Mad3/BubR1 protein is required during mitosis for the spindle assembly checkpoint (SAC). In meiosis, depletion causes an accelerated transit through prophase I and missegregation of achiasmate chromosomes in yeast [1], whereas in mice, reduced dosage leads to severe chromosome missegregation [2]. These observations indicate a meiotic requirement for BubR1, but its mechanism of action remains unknown. We identified a viable bubR1 allele in Drosophila resulting from a point mutation in the kinase domain that retains mitotic SAC activity. In males, we demonstrate a dose-sensitive requirement for BubR1 in maintaining sister-chromatid cohesion at anaphase I, whereas the mutant BubR1 protein localizes correctly. In bubR1 mutant females, we find that both achiasmate and chiasmate chromosomes nondisjoin mostly equationally consistent with a defect in sister-chromatid cohesion at late anaphase I or meiosis II. Moreover, mutations in bubR1 cause a consistent increase in pericentric heterochromatin exchange frequency, and although the synaptonemal complex is set up properly during transit through the germarium, it is disassembled prematurely in prophase by stage 1. Our results demonstrate that BubR1 is essential to maintain sister-chromatid cohesion during meiotic progression in both sexes and for normal maintenance of SC in females.

Figure 1. Meiotic Phenotype in bubR1 Mutant Males

(A) Nondisjunction frequency for the sex and the fourth chromosome among progeny from crosses between y/y⁺Y; bubR1^D1326N/bubR1^D1326N; spa^pol or y/y⁺Y; Df(2R)nap9/bubR1^D1326N; spa^pol males to y w sn; C(4)RM ci ey /0 females. (B) Quantification of the abnormal meiotic phenotypes in homozygous bubR1^D1326N males (n indicated in parentheses). A cell was scored as having PSCS at metaphase I, anaphase I, or metaphase II if sisters of at least one major chromosome were separate. Nondisjunction at anaphase I and II was scored if lagging chromosomes or unequal chromosome segregation was observed, and at telophase I and II if resulting sister nuclei were of different sizes or if micronuclei were present. (C–E) Abnormal meiotic phenotypes in homozygous bubR1^D1326N spermatocytes. (C) Late anaphase of meiosis I showing PSCS. (D) Metaphase II showing PSCS and three chromatids of the dot-like fourth chromosome, resulting from nondisjunction during meiosis I. (E) Anaphase II showing nondisjunction and a lagging chromosome. (F–I) BubR1 localization in homozygous spermatocytes during meiosis I and II. BubR1 is in red and DNA is in blue in all images. (F) Prophase I, (G) prometaphase I, (H) metaphase I, and (I) metaphase II. BubR1^D1326N shows a wild-type pattern of localization in mutant spermatocytes. Scale bar represents 10 µm.

Figure 2. Structure of the Synaptonemal Complex during the Transit through the Germarium and at Stage 1 Egg Chamber

(A–J) C(2)M is in red, C(3)G is in green, and DNA is in blue. (A and C–F) Wild-type SC within the germarium and at stage 1 of egg-chamber development. C(2)M and C(3)G colocalized in a well-defined structure along the DNA in the cells entering the meiotic cycle. During transit through the germarium, SC is restricted to the oocytes nucleus. At stage 1, only the oocyte nucleus retains a SC, as previously described [2]. (B and G–J) SC in Df(2R)nap9/bubR1^D1326N germarium and stage 1. While in region 2a, SC is formed within the cell entering the meiotic cycle; during the transit through the germarium, the SC fail to maintain and at stage 1, most of the C(2)M is dispersed within the oocyte and only partially colocalized with the DNA (n = 38/41). The C(3)G signal is reduced and partially colocalized with C(2)M while the DNA appears decondensed (n = 65/68). (K–N) Quantification of SC restriction and maintenance to the oocyte nucleus labeled with Orb during the transit through the germarium. In all images, C(2)M or C(3)G is in green, Orb is in red, and DNA is in blue. We analyzed by confocal microscopy each individual SC component (C(2)M, n = 32; and C(3)G, n = 36) during the transit through the germarium. In region 2a when the cysts enter meiosis, each SC component localized within 3 to 4 cells of the cyst. However, in region 2b, C(2)M (n = 30/32) appears dispersed within the cyst and is absent from the oocyte nucleus in region 3, while C(3)G (n = 36/36) signal is reduced. Scale bars represent 10 µm.

Figure 3. SMC1 Immunodetection during Oogenesis

In all images, SMC1 is in green, Orb is in red, and DNA is in blue. (A and B) SMC1 immunodetection in wild-type and BubR1mutant germarium. During the transit through the germarium, SMC1 accumulate within the oocyte nucleus detected by Orb (n = 12). In contrast, in BubR1 mutant germarium, SMC1 is strongly reduced within the oocyte nucleus (n = 14/14). (C–H) SMC1 immunodetection at stage 1 egg chamber. In wild-type egg chamber (n = 18), SMC1 localization is similar to SC, whereas in the BubR1 mutant egg chamber, SMC1 is diffused within the cytoplasm and only a low amount appears DNA bound (n = 18). (I–N) SMC1 immunodetection at stage 5. Whereas in both wild-type and BubR1mutant egg chamber, the karyosome appears normal when detected with DAPI, SMC1 localization differ. In wild-type, SMC1 localized in a well-defined pattern along the DNA (n = 16), whereas in the BubR1 mutant oocyte, only a low amount of SMC1 colocalized with DNA and SMC1 is not restricted into a well-defined structure. Scale bars represent 10 µm.

Figure 4. BubR1 Immunodetection during Oogenesis

In all images, BubR1 is in green, Orb is in red, and DNA is in blue. (A and B) BubR1 immunodetection in wild-type and mutant ovarioles. (C–F) Higher magnification of a germarium in both wild-type and mutant oocytes. In region 1 of both wild-type and mutant, BubR1 is detected within the mitotic cells, certainly representing BubR1 mitotic function. However, during the transit through the wild-type gemarium, BubR1is always observed to accumulate within the oocytes nucleus in region 3 as detected by Orb (n = 25). In contrast, in the mutant egg chamber, BubR1 mutant protein fails to accumulate within the oocyte in region 3 (n = 23/25). Higher magnification of a region 3 in (G, H) wild-type and (I, J) mutant germaria. BubR1 localized preferentially over the chromatin in wild-type oocytes, whereas the mutant protein is mostly dispersed. Scale bar represents 100 µm in (A) and (B) and 10 µm in (C)–(F).