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Regulation of centromere localization of the Drosophila Shugoshin MEI-S332 and sister-chromatid cohesion in meiosis.

Nogueira C, Kashevsky H, Pinto B, Clarke A, Orr-Weaver TL - G3 (Bethesda) (2014)

Bottom Line: These studies also suggested that MEI-S332 can be inactivated independently of delocalization, a conclusion supported here by localization and function studies in meiosis.Phosphoresistant and phosphomimetic mutants for the Aurora B and Polo phosphorylation sites were examined for effects on MEI-S332 localization and chromosome segregation in meiosis.Strikingly, MEI-S332 with a phosphomimetic mutation in the Aurora B phosphorylation site prematurely dissociates from the centromeres in meiosis I.

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Affiliation: Whitehead Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142.

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Dynamic distribution of MEI-S332 in male meiosis. (A) Localization of MEI-S332 through meiosis. Meiotic stages are labeled on the left: PM I, prometaphase I; M I, metaphase I; A I, anaphase I (early or late); T I, telophase I; P II, prophase II; PM II, prometaphase II; M II, metaphase II; A II, anaphase II (early or late); T II, telophase II. Merged panels show MEI-S332 antibody staining in green, tubulin in red, and DAPI in blue. Split channels are shown for MEI-S332 and DAPI. The dotted circles for the meiosis II panels demarcate individual spermatocytes. Scale bars = 10 μm. During metaphase I, MEI-S332 is detected enriched at the centromeres of the condensed chromosomes. By anaphase I, MEI-S332 is still found at centromeric dots but delocalizes from centromeres and becomes spread over the chromosome arms. Early and late anaphase I were distinguished by spindle morphology and the appearance of the spindle midzone. MEI-S332 is no longer detectable on chromosomes in late anaphase I. MEI-S332 is not present on chromosomes in telophase I and prophase II. In prometaphase II, MEI-S332 is detectable again at centromeres, persisting until early anaphase II. (B) Quantification of MEI-S332 localization. Blue indicates MEI-S332 observed solely in centromere foci, purple MEI-S332 detectable along the chromosomes and at the centromeres, and green no MEI-S332 visualized on the chromosomes. Numbers scored were: prometaphase I (n = 48), metaphase I (n = 28), anaphase I (n = 12), telophase I (n = 39), prophase II (n = 5), prometaphase II (n = 16), metaphase II (n = 18), anaphase II (n = 29), and telophase II (n = 65). The anaphase I bar includes both early and late anaphase I cells.
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fig1: Dynamic distribution of MEI-S332 in male meiosis. (A) Localization of MEI-S332 through meiosis. Meiotic stages are labeled on the left: PM I, prometaphase I; M I, metaphase I; A I, anaphase I (early or late); T I, telophase I; P II, prophase II; PM II, prometaphase II; M II, metaphase II; A II, anaphase II (early or late); T II, telophase II. Merged panels show MEI-S332 antibody staining in green, tubulin in red, and DAPI in blue. Split channels are shown for MEI-S332 and DAPI. The dotted circles for the meiosis II panels demarcate individual spermatocytes. Scale bars = 10 μm. During metaphase I, MEI-S332 is detected enriched at the centromeres of the condensed chromosomes. By anaphase I, MEI-S332 is still found at centromeric dots but delocalizes from centromeres and becomes spread over the chromosome arms. Early and late anaphase I were distinguished by spindle morphology and the appearance of the spindle midzone. MEI-S332 is no longer detectable on chromosomes in late anaphase I. MEI-S332 is not present on chromosomes in telophase I and prophase II. In prometaphase II, MEI-S332 is detectable again at centromeres, persisting until early anaphase II. (B) Quantification of MEI-S332 localization. Blue indicates MEI-S332 observed solely in centromere foci, purple MEI-S332 detectable along the chromosomes and at the centromeres, and green no MEI-S332 visualized on the chromosomes. Numbers scored were: prometaphase I (n = 48), metaphase I (n = 28), anaphase I (n = 12), telophase I (n = 39), prophase II (n = 5), prometaphase II (n = 16), metaphase II (n = 18), anaphase II (n = 29), and telophase II (n = 65). The anaphase I bar includes both early and late anaphase I cells.

Mentions: Two unexpected observations arose from anti-MEI-S332 staining of wild-type testes. First, although MEI-S332 is enriched at the centromere in early anaphase I, it is also detectable at lower levels throughout the chromosomes, revealing a redistribution at the metaphase I/anaphase I transition (Figure 1). Enhanced sensitivity of the anti-MEI-S332 antibody compared to the GFP signal used in previous studies likely accounts for our ability to observe the protein along the chromosome arms. The protein is not detectable on chromosomes in telophase I and prophase II but is localized to the centromeres in prometaphase II. The second new observation afforded by the antibody staining is that the protein is still present in the centromere region in some anaphase II cells. We do not observe it along chromosome arms as in early anaphase I cells. Because the protein does not delocalize completely at the metaphase II/anaphase II transition, there appears to be a mechanism to release cohesion without complete delocalization of MEI-S332, consistent with the cell culture studies.


Regulation of centromere localization of the Drosophila Shugoshin MEI-S332 and sister-chromatid cohesion in meiosis.

Nogueira C, Kashevsky H, Pinto B, Clarke A, Orr-Weaver TL - G3 (Bethesda) (2014)

Dynamic distribution of MEI-S332 in male meiosis. (A) Localization of MEI-S332 through meiosis. Meiotic stages are labeled on the left: PM I, prometaphase I; M I, metaphase I; A I, anaphase I (early or late); T I, telophase I; P II, prophase II; PM II, prometaphase II; M II, metaphase II; A II, anaphase II (early or late); T II, telophase II. Merged panels show MEI-S332 antibody staining in green, tubulin in red, and DAPI in blue. Split channels are shown for MEI-S332 and DAPI. The dotted circles for the meiosis II panels demarcate individual spermatocytes. Scale bars = 10 μm. During metaphase I, MEI-S332 is detected enriched at the centromeres of the condensed chromosomes. By anaphase I, MEI-S332 is still found at centromeric dots but delocalizes from centromeres and becomes spread over the chromosome arms. Early and late anaphase I were distinguished by spindle morphology and the appearance of the spindle midzone. MEI-S332 is no longer detectable on chromosomes in late anaphase I. MEI-S332 is not present on chromosomes in telophase I and prophase II. In prometaphase II, MEI-S332 is detectable again at centromeres, persisting until early anaphase II. (B) Quantification of MEI-S332 localization. Blue indicates MEI-S332 observed solely in centromere foci, purple MEI-S332 detectable along the chromosomes and at the centromeres, and green no MEI-S332 visualized on the chromosomes. Numbers scored were: prometaphase I (n = 48), metaphase I (n = 28), anaphase I (n = 12), telophase I (n = 39), prophase II (n = 5), prometaphase II (n = 16), metaphase II (n = 18), anaphase II (n = 29), and telophase II (n = 65). The anaphase I bar includes both early and late anaphase I cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: Dynamic distribution of MEI-S332 in male meiosis. (A) Localization of MEI-S332 through meiosis. Meiotic stages are labeled on the left: PM I, prometaphase I; M I, metaphase I; A I, anaphase I (early or late); T I, telophase I; P II, prophase II; PM II, prometaphase II; M II, metaphase II; A II, anaphase II (early or late); T II, telophase II. Merged panels show MEI-S332 antibody staining in green, tubulin in red, and DAPI in blue. Split channels are shown for MEI-S332 and DAPI. The dotted circles for the meiosis II panels demarcate individual spermatocytes. Scale bars = 10 μm. During metaphase I, MEI-S332 is detected enriched at the centromeres of the condensed chromosomes. By anaphase I, MEI-S332 is still found at centromeric dots but delocalizes from centromeres and becomes spread over the chromosome arms. Early and late anaphase I were distinguished by spindle morphology and the appearance of the spindle midzone. MEI-S332 is no longer detectable on chromosomes in late anaphase I. MEI-S332 is not present on chromosomes in telophase I and prophase II. In prometaphase II, MEI-S332 is detectable again at centromeres, persisting until early anaphase II. (B) Quantification of MEI-S332 localization. Blue indicates MEI-S332 observed solely in centromere foci, purple MEI-S332 detectable along the chromosomes and at the centromeres, and green no MEI-S332 visualized on the chromosomes. Numbers scored were: prometaphase I (n = 48), metaphase I (n = 28), anaphase I (n = 12), telophase I (n = 39), prophase II (n = 5), prometaphase II (n = 16), metaphase II (n = 18), anaphase II (n = 29), and telophase II (n = 65). The anaphase I bar includes both early and late anaphase I cells.
Mentions: Two unexpected observations arose from anti-MEI-S332 staining of wild-type testes. First, although MEI-S332 is enriched at the centromere in early anaphase I, it is also detectable at lower levels throughout the chromosomes, revealing a redistribution at the metaphase I/anaphase I transition (Figure 1). Enhanced sensitivity of the anti-MEI-S332 antibody compared to the GFP signal used in previous studies likely accounts for our ability to observe the protein along the chromosome arms. The protein is not detectable on chromosomes in telophase I and prophase II but is localized to the centromeres in prometaphase II. The second new observation afforded by the antibody staining is that the protein is still present in the centromere region in some anaphase II cells. We do not observe it along chromosome arms as in early anaphase I cells. Because the protein does not delocalize completely at the metaphase II/anaphase II transition, there appears to be a mechanism to release cohesion without complete delocalization of MEI-S332, consistent with the cell culture studies.

Bottom Line: These studies also suggested that MEI-S332 can be inactivated independently of delocalization, a conclusion supported here by localization and function studies in meiosis.Phosphoresistant and phosphomimetic mutants for the Aurora B and Polo phosphorylation sites were examined for effects on MEI-S332 localization and chromosome segregation in meiosis.Strikingly, MEI-S332 with a phosphomimetic mutation in the Aurora B phosphorylation site prematurely dissociates from the centromeres in meiosis I.

View Article: PubMed Central - PubMed

Affiliation: Whitehead Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142.

Show MeSH
Related in: MedlinePlus