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Figure 1. Characterization of Aurora localization during cleavage cycles and polar body extrusion.
A) i) Confocal images of the endogenous localization pattern of Aurora in P. mammillata embryos during one mitotic cycle. Aurora is localized in prophase to both centrosomes (blue arrows). At metaphase Aurora is strongly localized to both spindle poles (blue arrows) and weakly to the metaphase chromosomes (yellow arrow). At anaphase Aurora localizes to the spindle poles (blue arrows) and the spindle midzone (red arrow). At telophase Aurora localizes to the midbody (red arrows). Aurora was labeled with an anti-Aurora antibody (green), DAPI staining shows DNA/chromosomes (blue) and microtubules labeled with anti-tubulin antibody are depicted as red. Scale bars â=â10 µm. ii) GST-Aurora pull downs showing interaction between Ci-Aurora and Ci-TPX2 and Ci-INCENP. nâ=â3 experiments. B) Confocal images of live cleavage stage embryos showing Ci-Aurora::Venus localization pattern. i) A surface view of a 32 cell stage embryo shows that Aurora localizes strongly to the midbody (red arrows). Sister blastomeres (b6.7 and b6.8) are indicated on the right and left sides of this embryo (these embryos are bilaterally symmetrical). The boxed region has been enlarged to show the midbody labeling in greater detail. Confocal image of a neurula stage embryo showing Ci-Aurora::Venus localization to the centrosome (blue arrows), spindles (appear green), spindle poles (blue arrows) and the central spindle (red arrows). The boxed areas have been enlarged to highlight the centrosome, spindle and central spindle localization. Scale bar â=â50 µm. ii) Confocal images of Ci-Aurora::Venus (green) and histone H2B::Rfp1 (red) localization during one mitotic cycle in a cleavage stage embryo. Ci-Aurora::Venus localizes to both centrosomes (blue arrows), spindle poles (blue arrows), weakly to the chromosomes (yellow arrow), and the midbody (red arrow). Merged images are shown. Scale bar â=â10 µm. C) i) Confocal immunofluorescence images of endogenous Aurora (green), microtubules (red) and DNA (blue) stained with DAPI during first polar body extrusion. Aurora is enriched at the spindles poles (blue arrows), the chromosomes (yellow arrow), the spindle midzone (red arrow) and the midbody (red arrow). n â=â43 oocytes, 3 animals. Scale bars â=â10 µm. ii) Epifluorescence images showing Ci-Aurora::mCherry and Ci-Venus::INCENP localization through metaphase I to first polar body extrusion. Ci-Aurora::mCherry is localized on the metaphase I spindle (5 min.) then the midbody (9 min., red arrow). Ci-Venus::INCENP localization on metaphase I chromsomes (5 min., yellow arrow) then the midbody (9 min., red arrow). Corresponding bright field images showing polar body extrusion at 7 and 9 min. Merged fluorescence images showing midbody co-localization of Ci-Aurora::mCherry (red) and Ci-Venus::INCENP (red arrow). n â=â9 oocytes, 3 animals.
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Figure 2. Characterization of the midbody during maternal meiosis and mitosis.
A) Confocal images of TPX2, INCENP and Aurora immunofluorescence in Phallusia mammillata cleavage stage embryos. TPX2, INCENP and Aurora all label the midbody (red arrows) and all display a similar labeling pattern with a central dark zone at the centre of the midbody. Microtubules (red) and DNA/chromosomes (blue) labeled with DAPI are also shown. Scale bars â=â10 µm. B) Confocal images of TPX2 and INCENP immunofluorescence during extrusion of the first polar body. TPX2 labels the Meta I spindle and the midbody (red arrow). INCENP labels chromosomes at Meta I (yellow arrow) then the central spindle (red arrow) and midbody at Telophase I (red arrow). For INCENP the meiotic spindle was imaged in cross section in order to reveal INCENP kinetochore labeling of individual bivalent chromosomes more clearly. Scale bars â=â10 µm.
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Figure 3. Two different point mutations of Ci-Aurora prevent midbody localization.
A) Epifluorescence images from a 4D time-lapse series (time in min. is indicated). Point mutation of Ci-Aurora S60R::Venus prevents localization to the spindle pole as expected. However, midbody localization is also perturbed. Ci-Plk1::mCherry localization to the central spindle and midbody is indicated (red arrows) while Ci-Aurora S60R::Venus shows no midbody localization in the same cell. Corresponding brightfield images are shown. Scale barâ=â10 µm. nâ=â9, 3 animals. B) Epifluorescence images from a 4D time-lapse series. Point mutation of Ci-Aurora S60R::Venus prevents midbody localization. Ci-TPX2::mCherry localizes to the midbody (red arrow) while Ci-Aurora S60R::mCherry is completely absent from the midbody at telophase. Corresponding brightfield images are shown. Scale bar â=â10 µm. nâ=â9, 3 animals. C) Epifluorescence images from a time-lapse sequence showing that point mutation of Ci-Aurora G103N::Venus prevents midbody localization (grey circle) during telophase. Note that centrosome localization during interphase is preserved (blue arrows). HH2B::Rfp1 was used to precisely follow the mitotic cycle. Note also that AuroraG103N::Venus did not localize to the chromosomes. Corresponding brightfield images are shown. Scale bar â=â10 µm. nâ=â23, 3 animals. D) GST-CiAurwt, CiAurG103N and CiAurS60R were retained on the Sephadex beads for later interaction with radioactive 35S-labeled CiTPX2. More TPX2 was found attached to Aurwt than to either AurG103N or AurS60R. Three experiments.
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Figure 4. Inhibition of Aurora prevents first polar body extrusion.Unfertilized metaphase I arrested eggs were treated with 10 µM ZM447439 for 30 min. and then fertilized. A) Epifluorescence images of Ci-TPX2::Venus to label the spindle poles (blue arrows) and HH2B::Rfp1 to label the chromosomes. Following fertilization chromosomes segregate (4 min., HH2B::Rfp1 image) and the first polar body is extruded (7 min.) but is then re-absorbed (9 min.). Note also a common imaging arfefact in the H2B::Rfp image where there appears to be more H2B::Rfp on the cortex above the meiotic spindle at 4 min. This is likely caused by the curvature of the membrane and can be seen in most fluorescence images at this time. Corresponding brightfield images showing polar body extrusion and re-absorption. nâ=â17; 3 animals. B) Unfertilized metaphase I arrested eggs were treated with 10 µM ZM447439 for 30 min. and then fertilized. Metaphase I chromosomes labeled with Ci-INCENP::mCherry (yellow arrows). Following fertilization anaphase I occurs (7 min.) and the spindle appears positioned normally (7 min.). The first polar body is extruded but is then re-absorbed (10 min.) No Ci-INCENP::mCherry midbody labeling is present at this time. nâ=â9, 3 animals. All scale bars â=â10 µm. C) Histone H1 kinase activity (reflecting MPF activity) and MBP kinase activity (reflecting MAPK kinase activity) in control batches of eggs and in eggs bathed in 10 µM ZM447439. No difference in the activities of either kinase was detected. Three experiments.
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Figure 5. Ci-Aurora::Venus localization in sea urchins.Ci-Aurora::Venus and HH2B::Rfp1 mRNA were injected into unfertilized sea urchin eggs (Paracentrotus lividus) and the eggs subsequently fertilized. During cleavage cycles Ci-Aurora::Venus and HH2B::Rfp1 proteins are produced and become fluorescent. Confocal images from a time series of one mitotic cleavage cycle are depicted (at 0, 8, 18, 24 min.). Ci-Aurora::Venus localizes to the nucleus (green arrow), chromosomes (yellow arrow), the spindle pole (blue arrow) and finally to the central spindle and midbody (red arrow). Corresponding brightfield and merged images are shown (red is HH2B::Rfp1 and green Ci-Aurora::Venus). Scale bar is 10 µm. nâ=â16, 3 animals.
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Figure 6. Phylogenetic tree of deuterostome Aurora-A, Aurora-B and Aurora-AB kinases rooted by Plk1 kinases.Phylogenetic tree constructed using CLUSTALW in BioEdit to align the catalytic domains of deuterostome Auroras. The tree was produced using MEGA employing the neighbor joining method. Species represented :HsAur A and B (human), MsAur A and B (mouse), XlAur A and B (Xenopus laevis), DrAur A and B (Danio rerio), CiAur (Ciona intestinalis, ascidian), PmAur (Phallusia mammillata, ascidian), SpAur (Strongylocentrotus purpuratus, sea urchin), SkAur (Saccoglossus kowalevskii, hemichordate), BfAur (Branchiostoma floridae, cephalochordate), MgAur (Marthasterias glacialis, starfish), and PpAur (Patiria pectinifera, starfish). Human and Ciona Polo like kinase 1 were used as the outgroup.
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