Urochordate Ascidians Possess a Single Isoform of Aurora Kinase That Localizes to the Midbody via TPX2 in Eggs and Cleavage Stage Embryos

<div><p>Aurora kinases are key proteins found throughout the eukaryotes that control mitotic progression. Vertebrate Aurora-A and B kinases are thought to have evolved from a single Aurora-kinase isoform closest to that found in present day urochordates. In urochordate ascidians Aurora binds both TPX2 (a vertebrate AURKA partner) and INCENP (a vertebrate AURKB partner) and localizes to centrosomes and spindle microtubules as well as chromosomes and midbody during both meiosis and mitosis. Ascidian Aurora also displays this localization pattern during mitosis in echinoderms, strengthening the idea that non-vertebrate deuterostomes such as the urochordates and echinoderms possess a single form of Aurora kinase that has properties of vertebrate Aurora-kinase A and B. In the ascidian, TPX2 localizes to the centrosome and the spindle poles also as in vertebrates. However, we were surprised to find that TPX2 also localized strongly to the midbody in ascidian eggs and embryos. We thus examined more closely Aurora localization to the midbody by creating two separate point mutations of ascidian Aurora predicted to perturb binding to TPX2. Both forms of mutated Aurora behaved as predicted: neither localized to spindle poles where TPX2 is enriched. Interestingly, neither form of mutated Aurora localized to the midbody where TPX2 is also enriched, suggesting that ascidian Aurora midbody localization required TPX2 binding in ascidians. Functional analysis revealed that inhibition of Aurora kinase with a pharmacological inhibitor or with a dominant negative kinase dead form of Aurora caused cytokinesis failure and perturbed midbody formation during polar body extrusion. Our data support the view that vertebrate Aurora-A and B kinases evolved from a single non-vertebrate deuterostome ancestor. Moreover, since TPX2 localizes to the midbody in ascidian eggs and cleavage stage embryos it may be worthwhile re-assessing whether Aurora A kinase or TPX2 localize to the midbody in eggs and cleavage stage embryos.</p> </div

Inhibition of Aurora prevents first polar body extrusion.

<p>Unfertilized metaphase I arrested eggs were treated with 10 µM ZM447439 for 30 min. and then fertilized. <b>A</b>) 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. n = 17; 3 animals. <b>B</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. n = 9, 3 animals. All scale bars  = 10 µm. <b>C</b>) 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.</p

Phylogenetic tree of deuterostome Aurora-A, Aurora-B and Aurora-AB kinases rooted by Plk1 kinases.

<p>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 (<i>Xenopus laevis</i>), DrAur A and B (<i>Danio rerio</i>), CiAur (<i>Ciona intestinalis</i>, ascidian), PmAur (<i>Phallusia mammillata</i>, ascidian), SpAur (<i>Strongylocentrotus purpuratus</i>, sea urchin), SkAur (<i>Saccoglossus kowalevskii</i>, hemichordate), BfAur (<i>Branchiostoma floridae</i>, cephalochordate), MgAur (<i>Marthasterias glacialis</i>, starfish), and PpAur (<i>Patiria pectinifera</i>, starfish). Human and <i>Ciona</i> Polo like kinase 1 were used as the outgroup.</p

Two different point mutations of Ci-Aurora prevent midbody localization.

<p><b>A</b>) 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 bar = 10 µm. n = 9, 3 animals. <b>B</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  = 10 µm. n = 9, 3 animals. <b>C</b>) 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  = 10 µm. n = 23, 3 animals. D) GST-CiAur^wt, CiAur^G103N and CiAur^S60R were retained on the Sephadex beads for later interaction with radioactive ³⁵S-labeled CiTPX2. More TPX2 was found attached to Aur^wt than to either Aur^G103N or Aur^S60R. Three experiments.</p

Role of PB1 Midbody Remnant Creating Tethered Polar Bodies during Meiosis II

Polar body (PB) formation is an extreme form of unequal cell division that occurs in oocytes due to the eccentric position of the small meiotic spindle near the oocyte cortex. Prior to PB formation, a chromatin-centered process causes the cortex overlying the meiotic chromosomes to become polarized. This polarized cortical subdomain marks the site where a cortical protrusion or outpocket forms at the oocyte surface creating the future PBs. Using ascidians, we observed that PB1 becomes tethered to the fertilized egg via PB2, indicating that the site of PB1 cytokinesis directed the precise site for PB2 emission. We therefore studied whether the midbody remnant left behind following PB1 emission was involved, together with the egg chromatin, in defining the precise cortical site for PB2 emission. During outpocketing of PB2 in ascidians, we discovered that a small structure around 1 &micro;m in diameter protruded from the cortical outpocket that will form the future PB2, which we define as the &ldquo;polar corps&rdquo;. As emission of PB2 progressed, this small polar corps became localized between PB2 and PB1 and appeared to link PB2 to PB1. We tested the hypothesis that this small polar corps on the surface of the forming PB2 outpocket was the midbody remnant from the previous round of PB1 cytokinesis. We had previously discovered that Plk1::Ven labeled midbody remnants in ascidian embryos. We therefore used Plk1::Ven to follow the dynamics of the PB1 midbody remnant during meiosis II. Plk1::Ven strongly labeled the small polar corps that formed on the surface of the cortical outpocket that created PB2. Following emission of PB2, this polar corps was rich in Plk1::Ven and linked PB2 to PB1. By labelling actin (with TRITC-Phalloidin) we also demonstrated that actin accumulates at the midbody remnant and also forms a cortical cap around the midbody remnant in meiosis II that prefigured the precise site of cortical outpocketing during PB2 emission. Phalloidin staining of actin and immunolabelling of anti-phospho aPKC during meiosis II in fertilized eggs that had PB1 removed suggested that the midbody remnant remained within the fertilized egg following emission of PB1. Dynamic imaging of microtubules labelled with Ens::3GFP, MAP7::GFP or EB3::3GFP showed that one pole of the second meiotic spindle was located near the midbody remnant while the other pole rotated away from the cortex during outpocketing. Finally, we report that failure of the second meiotic spindle to rotate can lead to the formation of two cortical outpockets at anaphase II, one above each set of chromatids. It is not known whether the midbody remnant of PB1 is involved in directing the precise location of PB2 since our data are correlative in ascidians. However, a review of the literature indicates that PB1 is tethered to the egg surface via PB2 in several species including members of the cnidarians, lophotrochozoa and echinoids, suggesting that the midbody remnant formed during PB1 emission may be involved in directing the precise site of PB2 emission throughout the invertebrates

Release from meiotic arrest in ascidian eggs requires the activity of two phosphatases but not CaMKII

International audienceThe fertilising sperm triggers a transient Ca2+ increase that releases eggs from cell cycle arrest in the vast majority of animal eggs. In vertebrate eggs, Erp1, an APC/C-cdc20 inhibitor, links release from metaphase II arrest with the Ca2+ transient and its degradation is triggered by the Ca2+-induced activation of CaMKII. By contrast, many invertebrate groups have mature eggs that arrest at metaphase I, and these species do not possess the CaMKII target Erp1 in their genomes. As a consequence, it is unknown exactly how cell cycle arrest at metaphase I is achieved and how the fertilisation Ca2+ transient overcomes the arrest in the vast majority of animal species. Using live-cell imaging with a novel cyclin reporter to study cell cycle arrest and its release in urochordate ascidians, the closest living invertebrate group to the vertebrates, we have identified a new signalling pathway for cell cycle resumption in which CaMKII plays no part. Instead, we find that the Ca2+-activated phosphatase calcineurin (CN) is required for egg activation. Moreover, we demonstrate that parthenogenetic activation of metaphase I-arrested eggs by MEK inhibition, independent of a Ca2+ increase, requires the activity of a second egg phosphatase: PP2A. Furthermore, PP2A activity, together with CN, is required for normal egg activation during fertilisation. As ascidians are a sister group of the vertebrates, we discuss these findings in relation to cell cycle arrest and egg activation in chordates

Kif2 localizes to a subdomain of cortical endoplasmic reticulum that drives asymmetric spindle position

Early ascidian embryos have a cortical subdomain of endoplasmic reticulum (ER) that controls asymmetric spindle positioning driving unequal cell division. Here the authors show that the microtubule depolymerase Kif2 is localized to a cortical subdomain of the ER that is involved in asymmetric spindle positioning

High-content analysis of larval phenotypes for the screening of xenobiotic toxicity using Phallusia mammillata embryos

International audienceIn recent years, pollution of surface waters with xenobiotic compounds became an issue of concern in society and has been the object of numerous studies. Most of these xenobiotic compounds are man-made molecules and some of them are qualified as endocrine disrupting chemicals (EDCs) when they interfere with hormones actions. Several studies have investigated the teratogenic impacts of EDCs in vertebrates (including marine vertebrates). However, the impact of such EDCs on marine invertebrates is much debated and still largely obscure. In addition, DNA-altering genotoxicants can induce embryonic malformations. The goal of this study is to develop a reliable and effective test for assessing toxicity of chemicals using embryos of the ascidian (Phallusia mammillata) in order to find phenotypic signatures associated with xenobiotics. We evaluated embryonic malformations with high-content analysis of larval phenotypes by scoring several quantitative and qualitative morphometric endpoints on a single image of Phallusia tadpole larvae with semi-automated image analysis. Using this approach we screened different classes of toxicants including genotoxicants, known or suspected EDCs and nuclear receptors (NRs) ligands. The screen presented here reveals a specific phenotypic signature for ligands of retinoic acid receptor/retinoid X receptor. Analysis of larval morphology combined with DNA staining revealed that embryos with DNA aberrations displayed severe malformations affecting multiple aspects of embryonic development. In contrast EDCs exposure induced no or little DNA aberrations and affected mainly neural development. Therefore the ascidian embryo/larval assay presented here can allow to distinguish the type of teratogenicity induced by different classes of toxicants