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. 2006 Jul;11(1):57-68.
doi: 10.1016/j.devcel.2006.04.021.

INCENP and Aurora B promote meiotic sister chromatid cohesion through localization of the Shugoshin MEI-S332 in Drosophila

Tamar D Resnick  1 , David L Satinover, Fiona MacIsaac, P Todd Stukenberg, William C Earnshaw, Terry L Orr-Weaver, Mar Carmena
Affiliations

INCENP and Aurora B promote meiotic sister chromatid cohesion through localization of the Shugoshin MEI-S332 in Drosophila

Tamar D Resnick et al. Dev Cell. 2006 Jul.

Abstract

The chromosomal passenger complex protein INCENP is required in mitosis for chromosome condensation, spindle attachment and function, and cytokinesis. Here, we show that INCENP has an essential function in the specialized behavior of centromeres in meiosis. Mutations affecting Drosophila incenp profoundly affect chromosome segregation in both meiosis I and II, due, at least in part, to premature sister chromatid separation in meiosis I. INCENP binds to the cohesion protector protein MEI-S332, which is also an excellent in vitro substrate for Aurora B kinase. A MEI-S332 mutant that is only poorly phosphorylated by Aurora B is defective in localization to centromeres. These results implicate the chromosomal passenger complex in directly regulating MEI-S332 localization and, therefore, the control of sister chromatid cohesion in meiosis.

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Figures

Figure 1
Figure 1. INCENP Protein Localization and Mutant Defects in Meiosis I
(A) Wild-type metaphase I: INCENP concentrated on centromeres (arrow). (B) Wild-type late anaphase I: INCENP remains on centromeres, and some protein transfers to the central spindle (arrow). (C) P(EP)2340 prometaphase I: abnormally condensed bivalents, an abnormally long and wavy spindle, decreased levels of centromeric INCENP. (D) Same as (C), but INCENP staining is undetectable. (E) QA26 meiosis I: INCENP on centromeres and small segments of chromatin, the result of chromosome fragmentation or aberrant condensation. Scale bars are 5 μm.
Figure 2
Figure 2. INCENP Protein Localization and Mutant Defects in Meiosis II
(A) Wild-type anaphase II: INCENP associated with chromatin, central spindle, and cell cortex (arrow). (B) P(EP)2340 prometaphase II-like figure: elongated spindle, decreased levels of centromeric INCENP, and chromatin masses aligned along the spindle. (C) Meiosis II spindle showing the absence of INCENP staining and missegregation of chromosome 4. (D) QA26 prometaphase II-like figure showing the absence of INCENP from some chromosomes and both copies of chromosome 4 at one pole. Scale bars are 5 μm.
Figure 3
Figure 3. Meiotic Chromosome Cohesion and Condensation Defects in incenp Mutants
(A) Mutations in incenp cause elevated nondisjunction during meiotic chromosome segregation. (B) Defective prophase I chromosome condensation in a QA26 mutant spermatocyte. (C) QA26 mutant prophase I: chromatid arms protrude from loosely packed bivalents (arrow). The inset shows the wild-type prophase I bivalent configuration. (D) Wild-type anaphase I: at one pole, one major autosome (arrow) and the X chromosome (arrowhead) attached at their centromeres are indicated. This cohesion configuration is retained into prometaphase II. (E) Anaphase I QA26 mutant: sister chromatids of all dyads at one pole (arrow) are precociously separated. (F) Prometaphase II QA26 spermatocyte: sister chromatids of one dyad have lost cohesion (arrow) rather than retaining cohesion at the centromere, as seen in the adjacent dyad (arrowhead). (G) Aneuploid prometaphase II QA26 mutant cell with more than the six expected major chromatids (see [D] and [F]), which is most likely the result of meiosis I nondisjunction. Scale bars are 10 μm.
Figure 4
Figure 4. Loss of INCENP/Aurora B in Mitosis Correlates with Delocalization of MEI-S332
(A) Distribution of mitotic phases in DmIN-CENP RNAi (red bar), DmAurora B RNAi (yellow bar), and control (blue bar) shows a reduction of the percentage of metaphases and an increase in the percentage of prometaphases (t = 48 hr after addition of dsRNA); the percentage of abnormal anaphase cells is shown separately (right). (B) Analysis of the colocalization of MEI-S332 with INCENP in control and INCENP RNAi-treated cells (upper panel) and with Aurora B in Aurora B RNAi-treated cells (lower panel) (t = 48 hr). n > 300. (C) Localization of MEI-S332 and INCENP in control S2 cells. The zoomed image at right shows that both proteins overlap partially, but that INCENP extends beneath MEI-S332. The scale bar is 5 μm. (D) INCENP dsRNA-treated S2 cells showing unaligned chromosomes. INCENP and MEI-S332 are present on most centromeres. The Arrow points to a chromosome in which both proteins are absent. (E) INCENP dsRNA-treated S2 cells showing the absence of both INCENP and MEI-S332 (arrow). The scale bar is 5 μm. (F-I) Aurora B dsRNA-treated S2 cells. (F) Control metaphase cell; the zoomed image shows partial colocalization of Aurora B and MEI-S332 on centromeres. (G) Prometaphase cell with decondensed chromosomes showing the absence of both Aurora B and MEI-S332 from centromeres. (H) Prometaphase cell showing INCENP and MEI-S332 on some centromeres; the arrow points to an unaligned chromosome showing low levels of INCENP and undetectable MEI-S332. The scale baris 5 mm. (I) Prometaphase cell showing abnormal INCENP localization on chromatin and dispersed MEI-S332 staining.
Figure 5
Figure 5. Localization of INCENP and MEI-S332 Is Disrupted in QA26 Meiosis
(A) Wild-type metaphase I: INCENP and MEI-S332 on centromeres. The inset shows INCENP staining partially overlapping and linking the two sister kinetochore-associated MEI-S332 dots. (B) Wild-type early anaphase I: INCENP and MEI-S332 remain associated with the centromeres. The inset shows an overlap in the localization of the proteins. Arrow: absence of INCENP staining on the central spindle. (C) Wild-type late anaphase I: INCENP remains associated with centromeres, but some protein is associated with chromatin and the central spindle (arrow: INCENP on the central spindle). (D) QA26 meiosis I spermatocyte: INCENP and MEI-S332 are distributed diffusely on the chromosomes. (E) QA26 anaphase I: both INCENP and MEI-S332 are absent from the chromosomes. Scale bars are 5 μm.
Figure 6
Figure 6. INCENP Binds MEI-S332 In Vitro
(A) Proteins were translated in the presence of [35S]methionine and incubated with bacterially expressed GST-DmINCENP or GST bound to glutathione Sepharose beads. Bound (“B”) and unbound (“U”) fractions were separated by SDS-PAGE, and proteins were visualized by using a phosphorimager. (B) Quantification of the binding experiment shown in (A). The bars represent the percentage of total protein bound to GST (black) or GST-DmINCENP (gray). Error bars show standard deviation.
Figure 7
Figure 7. Aurora B Phosphorylates MEI-S332 and Regulates Its Stable Association with Centromeres in Mitosis
(A and B) INCENP/Aurora B phosphorylates MEI-S332 in vitro within residues 124–126 (A) The recombinant INCENP/Aurora B complex was incubated with [32P]ATP and the indicated substrate for 1 min, and incorporation of phosphate onto the proteins was visualized by autoradiography(right) and protein loading analyzed by Coomassie blue(left). MBP, myelin basic protein; GST, glutathioneS-transferase. (B) Timecourse of INCENP/Aurora B kinase activity (assayed as in [A]) by using wild-type MEI-S332 or the indicated phosphosite mutant. (C–G) The MEI-S332-124AAA phosphorylation mutant does not stably associate with centromeres in mitosis. (C) High level of centromeric GFP-MEI-S332 in metaphase. (D) Reduced level of the phosphorylation mutant GFP-MEI-S332-124AAA on metaphase centromeres (arrow). (E) Microscope field showing a prometaphase cell with high levels of centromeric GFP-MEI-S332-124AAA and a metaphase cell with very reduced levels of mutant protein in most centromeres (arrow). In (C)–(E), the GFP staining alone is shown in gray. (F) Western blot showing levels of expression of the GFP-tagged proteins in three different transfection experiments. (G) Percentage of cells transfected with GFP-MEI-S332 or GFP-MEI-S332-124AAA showing normal levels of GFP signal on kinetochores (HIGH), lower than normal levels (LOW), or no signal (NEGATIVE). Error bars show standard deviation. (H) Model of the role of INCENP in the regulation of MEI-S332 in meiosis. Left column: meiotic chromosome dynamics and the localization patterns of key regulatory proteins INCENP/Aurora B (orange/pink), MEI-S332 (green), and POLO (red). Right column: protein interactions at the kinetochore. In prophase I, CDK phosphorylates INCENP at the POLO binding site, promoting the targeting of POLO kinase to the kinetochore; INCENP targets Aurora B to the kinetochore; MEI-S332 is recruited to the kinetochore. Before the metaphase-anaphase I transition, Aurora B initiates its autoactivation backloop, phosphorylating INCENP and itself. The INCENP/Aurora B complex stabilizes centromeric MEI-S332 through direct binding and phosphorylation. At the onset of anaphase I, INCENP stays on the centromere, stopping MEI-S332 from being phosphorylated by POLO, and POLO transfers to the central spindle. During the metaphase-anaphase II transition, INCENP transitions off the centromere, redistributing over chromatin and transferring to the central spindle. POLO is free to phosphorylate MEI-S332, promoting its release from centromeres. POLO then transfers to the central spindle.
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References

    1. Adams RR, Tavares AA, Salzberg A, Bellen HJ, Glover DM. pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis. Genes Dev. 1998;12:1483–1494. - PMC - PubMed
    1. Adams RR, Wheatley SP, Gouldsworthy AM, Kandels-Lewis SE, Carmena M, Smythe C, Gerloff DL, Earnshaw WC. INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow. Curr Biol. 2000;10:1075–1078. - PubMed
    1. Adams RR, Eckley DM, Vagnarelli P, Wheatley SP, Gerloff DL, Mackay AM, Svingen PA, Kaufmann SH, Earnshaw WC. Human INCENP colocalizes with the Aurora-B/AIRK2 kinase on chromosomes and is overexpressed in tumour cells. Chromosoma. 2001a;110:65–74. - PubMed
    1. Adams RR, Maiato H, Earnshaw WC, Carmena M. Essential roles of Drosophila innercentromere protein (INCENP) and Aurora B in histone H3 phosphorylation, metaphase chromosome alignment, kinetochore disjunction, and chromosome segregation. J Cell Biol. 2001b;153:865–880. - PMC - PubMed
    1. Ainsztein AM, Kandels-Lewis SE, Mackay AM, Earnshaw WC. INCENP centromere and spindle targeting: identification of essential conserved motifs and involvement of heterochromatin protein HP1. J Cell Biol. 1998;143:1763–1774. - PMC - PubMed

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