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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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Localization of a MEI-S332 phosphorylation mimic in a POLO binding site (MEI-S332T331-D) during male meiosis. (A) Localization of MEI-S332T331-D in meiosis I and II. Labels and scale bars as in Figure 1. The dotted lines highlight individual metaphase I or II spermatocytes, except two metaphase II spermatocytes are included in the large, bottom circle of the metaphase II panel. The arrow in the metaphase I panel shows a spermatocyte with MEI-S332T331-D coating the chromosome arms, whereas the asterisk shows localization specifically to the centromeres. An early anaphase I spermatocyte is shown. All three metaphase II figures shown have MEI-S332T331D all along the chromosomes (arrow in the top nucleus and asterisks in bottom circle containing two metaphase II figures). Puncta of MEI-S332-GFP are present in the cytoplasm of the meiosis I spermatocytes. (B) Quantification of MEI-S332T331-D localization in metaphase I (n = 30), anaphase I (n = 63), metaphase II (n = 24), and telophase II (n = 457). Blue indicates MEI-S332 present solely at centromeres, red shows localization along the chromosome arms, and green indicates no detectable MEI-S332.
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fig5: Localization of a MEI-S332 phosphorylation mimic in a POLO binding site (MEI-S332T331-D) during male meiosis. (A) Localization of MEI-S332T331-D in meiosis I and II. Labels and scale bars as in Figure 1. The dotted lines highlight individual metaphase I or II spermatocytes, except two metaphase II spermatocytes are included in the large, bottom circle of the metaphase II panel. The arrow in the metaphase I panel shows a spermatocyte with MEI-S332T331-D coating the chromosome arms, whereas the asterisk shows localization specifically to the centromeres. An early anaphase I spermatocyte is shown. All three metaphase II figures shown have MEI-S332T331D all along the chromosomes (arrow in the top nucleus and asterisks in bottom circle containing two metaphase II figures). Puncta of MEI-S332-GFP are present in the cytoplasm of the meiosis I spermatocytes. (B) Quantification of MEI-S332T331-D localization in metaphase I (n = 30), anaphase I (n = 63), metaphase II (n = 24), and telophase II (n = 457). Blue indicates MEI-S332 present solely at centromeres, red shows localization along the chromosome arms, and green indicates no detectable MEI-S332.

Mentions: A phosphomimetic version of the Polo box-binding domain was made in which T331 was replaced with aspartic acid (T331-D). In vitro binding studies with Polo (see File S1) showed that this form of MEI-S332 exhibited enhanced binding to Polo (Figure S3). The behavior of MEI-S332-GFPT331-D was examined in transgenic lines as for the other MEI-S332 phosphomutants (Figure 5). In metaphase I, the protein localized to centromeres in 60% of spermatocytes, but in the remaining spermatocytes, weak arm localization was present. The majority of anaphase I cells lacked detectable MEI-S332 on the chromosomes. Nearly all metaphase II cells showed weak staining for MEI-S332 along the chromosomes. Thus, the T331-D mutation reduces centromere localization of MEI-S332, instead there are low levels of protein binding along the chromosome arms.


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)

Localization of a MEI-S332 phosphorylation mimic in a POLO binding site (MEI-S332T331-D) during male meiosis. (A) Localization of MEI-S332T331-D in meiosis I and II. Labels and scale bars as in Figure 1. The dotted lines highlight individual metaphase I or II spermatocytes, except two metaphase II spermatocytes are included in the large, bottom circle of the metaphase II panel. The arrow in the metaphase I panel shows a spermatocyte with MEI-S332T331-D coating the chromosome arms, whereas the asterisk shows localization specifically to the centromeres. An early anaphase I spermatocyte is shown. All three metaphase II figures shown have MEI-S332T331D all along the chromosomes (arrow in the top nucleus and asterisks in bottom circle containing two metaphase II figures). Puncta of MEI-S332-GFP are present in the cytoplasm of the meiosis I spermatocytes. (B) Quantification of MEI-S332T331-D localization in metaphase I (n = 30), anaphase I (n = 63), metaphase II (n = 24), and telophase II (n = 457). Blue indicates MEI-S332 present solely at centromeres, red shows localization along the chromosome arms, and green indicates no detectable MEI-S332.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4199692&req=5

fig5: Localization of a MEI-S332 phosphorylation mimic in a POLO binding site (MEI-S332T331-D) during male meiosis. (A) Localization of MEI-S332T331-D in meiosis I and II. Labels and scale bars as in Figure 1. The dotted lines highlight individual metaphase I or II spermatocytes, except two metaphase II spermatocytes are included in the large, bottom circle of the metaphase II panel. The arrow in the metaphase I panel shows a spermatocyte with MEI-S332T331-D coating the chromosome arms, whereas the asterisk shows localization specifically to the centromeres. An early anaphase I spermatocyte is shown. All three metaphase II figures shown have MEI-S332T331D all along the chromosomes (arrow in the top nucleus and asterisks in bottom circle containing two metaphase II figures). Puncta of MEI-S332-GFP are present in the cytoplasm of the meiosis I spermatocytes. (B) Quantification of MEI-S332T331-D localization in metaphase I (n = 30), anaphase I (n = 63), metaphase II (n = 24), and telophase II (n = 457). Blue indicates MEI-S332 present solely at centromeres, red shows localization along the chromosome arms, and green indicates no detectable MEI-S332.
Mentions: A phosphomimetic version of the Polo box-binding domain was made in which T331 was replaced with aspartic acid (T331-D). In vitro binding studies with Polo (see File S1) showed that this form of MEI-S332 exhibited enhanced binding to Polo (Figure S3). The behavior of MEI-S332-GFPT331-D was examined in transgenic lines as for the other MEI-S332 phosphomutants (Figure 5). In metaphase I, the protein localized to centromeres in 60% of spermatocytes, but in the remaining spermatocytes, weak arm localization was present. The majority of anaphase I cells lacked detectable MEI-S332 on the chromosomes. Nearly all metaphase II cells showed weak staining for MEI-S332 along the chromosomes. Thus, the T331-D mutation reduces centromere localization of MEI-S332, instead there are low levels of protein binding along the chromosome arms.

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