Publications by authors named "Katja Wassmann"

29 Publications

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Kinetochore individualization in meiosis I is required for centromeric cohesin removal in meiosis II.

EMBO J 2021 Mar 1:e106797. Epub 2021 Mar 1.

Institut de Biologie Paris Seine, Sorbonne Université, Paris, France.

Partitioning of the genome in meiosis occurs through two highly specialized cell divisions, named meiosis I and meiosis II. Step-wise cohesin removal is required for chromosome segregation in meiosis I, and sister chromatid segregation in meiosis II. In meiosis I, mono-oriented sister kinetochores appear as fused together when examined by high-resolution confocal microscopy, whereas they are clearly separated in meiosis II, when attachments are bipolar. It has been proposed that bipolar tension applied by the spindle is responsible for the physical separation of sister kinetochores, removal of cohesin protection, and chromatid separation in meiosis II. We show here that this is not the case, and initial separation of sister kinetochores occurs already in anaphase I independently of bipolar spindle forces applied on sister kinetochores, in mouse oocytes. This kinetochore individualization depends on separase cleavage activity. Crucially, without kinetochore individualization in meiosis I, bivalents when present in meiosis II oocytes separate into chromosomes and not sister chromatids. This shows that whether centromeric cohesin is removed or not is determined by the kinetochore structure prior to meiosis II.
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http://dx.doi.org/10.15252/embj.2020106797DOI Listing
March 2021

A PP2A-B56-Centered View on Metaphase-to-Anaphase Transition in Mouse Oocyte Meiosis I.

Cells 2020 02 7;9(2). Epub 2020 Feb 7.

Mammalian Oocyte Meiosis (MOM) UMR7622, Institut de Biologie Paris Seine, Sorbonne Université, 75005 Paris, France.

Meiosis is required to reduce to haploid the diploid genome content of a cell, generating gametes-oocytes and sperm-with the correct number of chromosomes. To achieve this goal, two specialized cell divisions without intermediate S-phase are executed in a time-controlled manner. In mammalian female meiosis, these divisions are error-prone. Human oocytes have an exceptionally high error rate that further increases with age, with significant consequences for human fertility. To understand why errors in chromosome segregation occur at such high rates in oocytes, it is essential to understand the molecular players at work controlling these divisions. In this review, we look at the interplay of kinase and phosphatase activities at the transition from metaphase-to-anaphase for correct segregation of chromosomes. We focus on the activity of PP2A-B56, a key phosphatase for anaphase onset in both mitosis and meiosis. We start by introducing multiple roles PP2A-B56 occupies for progression through mitosis, before laying out whether or not the same principles may apply to the first meiotic division in oocytes, and describing the known meiosis-specific roles of PP2A-B56 and discrepancies with mitotic cell cycle regulation.
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http://dx.doi.org/10.3390/cells9020390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072534PMC
February 2020

Cycling through mammalian meiosis: B-type cyclins in oocytes.

Cell Cycle 2019 07 23;18(14):1537-1548. Epub 2019 Jun 23.

a Institut de Biologie Paris Seine (IBPS) , Sorbonne Université , Paris , France.

B-type cyclins in association with Cdk1 mediate key steps of mitosis and meiosis, by phosphorylating a plethora of substrates. Progression through the meiotic cell cycle requires the execution of two cell divisions named meiosis I and II without intervening S-phase, to obtain haploid gametes. These two divisions are highly asymmetric in the large oocyte. Chromosome segregation in meiosis I and sister chromatid segregation in meiosis II requires the sharp, switch-like inactivation of Cdk1 activity, which is brought about by degradation of B-type cyclins and counteracting phosphatases. Importantly and contrary to mitosis, inactivation of Cdk1 must not allow S-phase to take place at exit from meiosis I. Here, we describe recent studies on the regulation of translation and degradation of B-type cyclins in mouse oocytes, and how far their roles are redundant or specific, with a special focus on the recently discovered oocyte-specific role of cyclin B3.
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http://dx.doi.org/10.1080/15384101.2019.1632139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6619999PMC
July 2019

Cyclin B3 promotes anaphase I onset in oocyte meiosis.

J Cell Biol 2019 04 5;218(4):1265-1281. Epub 2019 Feb 5.

Institut de Biologie Paris Seine, Sorbonne Université, Paris, France

Meiosis poses unique challenges because two rounds of chromosome segregation must be executed without intervening DNA replication. Mammalian cells express numerous temporally regulated cyclins, but how these proteins collaborate to control meiosis remains poorly understood. Here, we show that female mice genetically ablated for cyclin B3 are viable-indicating that the protein is dispensable for mitotic divisions-but are sterile. Mutant oocytes appear normal until metaphase I but then display a highly penetrant failure to transition to anaphase I. They arrest with hallmarks of defective anaphase-promoting complex/cyclosome (APC/C) activation, including no separase activity, high CDK1 activity, and high cyclin B1 and securin levels. Partial APC/C activation occurs, however, as exogenously expressed APC/C substrates can be degraded. Cyclin B3 forms active kinase complexes with CDK1, and meiotic progression requires cyclin B3-associated kinase activity. Cyclin B3 homologues from frog, zebrafish, and fruit fly rescue meiotic progression in cyclin B3-deficient mouse oocytes, indicating conservation of the biochemical properties and possibly cellular functions of this germline-critical cyclin.
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http://dx.doi.org/10.1083/jcb.201808091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446836PMC
April 2019

Detection of Separase Activity Using a Cleavage Sensor in Live Mouse Oocytes.

Methods Mol Biol 2018 ;1818:99-112

Sorbonne Universités, UPMC Université Paris 06, Institut de Biologie Paris Seine (IBPS), UMR7622, Paris, France.

Separase proteolytically removes cohesin complexes from sister chromatid arms in meiosis I, which is essential for chromosome segregation. Regulation of separase activity is essential for proper cell cycle progression and correct chromosome segregation. Onset of endogenous separase activity has not yet been observed in live oocytes.We describe here a method for detecting separase activity in mouse oocytes in vivo. This method utilizes a previously described cleavage sensor made up of H2B-mCherry fused with Scc1(107-268 aa)-YFP. The cleavage sensor is loaded on the chromosomes through its H2B-tag, and the signal from both mCherry and YFP is visible. Upon separase activation the Scc1 fragment is cleaved and YFP dissociates from the chromosomes. The change in the ratio between mCherry and YFP fluorescence intensity is a readout of separase activity.
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http://dx.doi.org/10.1007/978-1-4939-8603-3_11DOI Listing
March 2019

Multiple Duties for Spindle Assembly Checkpoint Kinases in Meiosis.

Front Cell Dev Biol 2017 13;5:109. Epub 2017 Dec 13.

Sorbonne Universités, UPMC Univ Paris 06, Institut de Biologie Paris Seine, UMR7622, Paris, France.

Cell division in mitosis and meiosis is governed by evolutionary highly conserved protein kinases and phosphatases, controlling the timely execution of key events such as nuclear envelope breakdown, spindle assembly, chromosome attachment to the spindle and chromosome segregation, and cell cycle exit. In mitosis, the spindle assembly checkpoint (SAC) controls the proper attachment to and alignment of chromosomes on the spindle. The SAC detects errors and induces a cell cycle arrest in metaphase, preventing chromatid separation. Once all chromosomes are properly attached, the SAC-dependent arrest is relieved and chromatids separate evenly into daughter cells. The signaling cascade leading to checkpoint arrest depends on several protein kinases that are conserved from yeast to man. In meiosis, haploid cells containing new genetic combinations are generated from a diploid cell through two specialized cell divisions. Though apparently less robust, SAC control also exists in meiosis. Recently, it has emerged that SAC kinases have additional roles in executing accurate chromosome segregation during the meiotic divisions. Here, we summarize the main differences between mitotic and meiotic cell divisions, and explain why meiotic divisions pose special challenges for correct chromosome segregation. The less-known meiotic roles of the SAC kinases are described, with a focus on two model systems: yeast and mouse oocytes. The meiotic roles of the canonical checkpoint kinases Bub1, Mps1, the pseudokinase BubR1 (Mad3), and Aurora B and C (Ipl1) will be discussed. Insights into the molecular signaling pathways that bring about the special chromosome segregation pattern during meiosis will help us understand why human oocytes are so frequently aneuploid.
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http://dx.doi.org/10.3389/fcell.2017.00109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733479PMC
December 2017

Tension-Induced Error Correction and Not Kinetochore Attachment Status Activates the SAC in an Aurora-B/C-Dependent Manner in Oocytes.

Curr Biol 2018 01 21;28(1):130-139.e3. Epub 2017 Dec 21.

Sorbonne Universités, UPMC Univ. Paris 06, CNRS FRE3631, Institut de Biologie Paris Seine (IBPS), 7 Quai St. Bernard, Paris 75005, France; CNRS UMR7622, Developmental Biology Lab, 7 Quai St. Bernard, 75005 Paris, France. Electronic address:

Cell division with partitioning of the genetic material should take place only when paired chromosomes named bivalents (meiosis I) or sister chromatids (mitosis and meiosis II) are correctly attached to the bipolar spindle in a tension-generating manner. For this to happen, the spindle assembly checkpoint (SAC) checks whether unattached kinetochores are present, in which case anaphase onset is delayed to permit further establishment of attachments. Additionally, microtubules are stabilized when they are attached and under tension. In mitosis, attachments not under tension activate the so-named error correction pathway depending on Aurora B kinase substrate phosphorylation. This leads to microtubule detachments, which in turn activates the SAC [1-3]. Meiotic divisions in mammalian oocytes are highly error prone, with severe consequences for fertility and health of the offspring [4, 5]. Correct attachment of chromosomes in meiosis I leads to the generation of stretched bivalents, but-unlike mitosis-not to tension between sister kinetochores, which co-orient. Here, we set out to address whether reduction of tension applied by the spindle on bioriented bivalents activates error correction and, as a consequence, the SAC. Treatment of oocytes in late prometaphase I with Eg5 kinesin inhibitor affects spindle tension, but not attachments, as we show here using an optimized protocol for confocal imaging. After Eg5 inhibition, bivalents are correctly aligned but less stretched, and as a result, Aurora-B/C-dependent error correction with microtubule detachment takes place. This loss of attachments leads to SAC activation. Crucially, SAC activation itself does not require Aurora B/C kinase activity in oocytes.
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http://dx.doi.org/10.1016/j.cub.2017.11.049DOI Listing
January 2018

Mps1 kinase-dependent Sgo2 centromere localisation mediates cohesin protection in mouse oocyte meiosis I.

Nat Commun 2017 09 25;8(1):694. Epub 2017 Sep 25.

Sorbonne Universités, UPMC Univ. Paris 06, Institut de Biologie Paris Seine (IBPS), UMR7622, Paris, 75005, France.

A key feature of meiosis is the step-wise removal of cohesin, the protein complex holding sister chromatids together, first from arms in meiosis I and then from the centromere region in meiosis II. Centromeric cohesin is protected by Sgo2 from Separase-mediated cleavage, in order to maintain sister chromatids together until their separation in meiosis II. Failures in step-wise cohesin removal result in aneuploid gametes, preventing the generation of healthy embryos. Here, we report that kinase activities of Bub1 and Mps1 are required for Sgo2 localisation to the centromere region. Mps1 inhibitor-treated oocytes are defective in centromeric cohesin protection, whereas oocytes devoid of Bub1 kinase activity, which cannot phosphorylate H2A at T121, are not perturbed in cohesin protection as long as Mps1 is functional. Mps1 and Bub1 kinase activities localise Sgo2 in meiosis I preferentially to the centromere and pericentromere respectively, indicating that Sgo2 at the centromere is required for protection.In meiosis I centromeric cohesin is protected by Sgo2 from Separase-mediated cleavage ensuring that sister chromatids are kept together until their separation in meiosis II. Here the authors demonstrate that Bub1 and Mps1 kinase activities are required for Sgo2 localisation to the centromere region.
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http://dx.doi.org/10.1038/s41467-017-00774-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612927PMC
September 2017

Meiotic Divisions: No Place for Gender Equality.

Adv Exp Med Biol 2017 ;1002:1-17

Sorbonne Universités, UPMC Univ Paris 06, Institut de Biologie Paris Seine (IBPS), UMR7622, Paris, 75252, France.

In multicellular organisms the fusion of two gametes with a haploid set of chromosomes leads to the formation of the zygote, the first cell of the embryo. Accurate execution of the meiotic cell division to generate a female and a male gamete is required for the generation of healthy offspring harboring the correct number of chromosomes. Unfortunately, meiosis is error prone. This has severe consequences for fertility and under certain circumstances, health of the offspring. In humans, female meiosis is extremely error prone. In this chapter we will compare male and female meiosis in humans to illustrate why and at which frequency errors occur, and describe how this affects pregnancy outcome and health of the individual. We will first introduce key notions of cell division in meiosis and how they differ from mitosis, followed by a detailed description of the events that are prone to errors during the meiotic divisions.
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http://dx.doi.org/10.1007/978-3-319-57127-0_1DOI Listing
September 2017

Super-resolution for everybody: An image processing workflow to obtain high-resolution images with a standard confocal microscope.

Methods 2017 02 5;115:17-27. Epub 2016 Nov 5.

Sorbonne Universités, UPMC Univ Paris 06, CNRS, Core Facilities - Institut de Biologie Paris Seine (IBPS), 75005 Paris, France. Electronic address:

In the presented work we aimed at improving confocal imaging to obtain highest possible resolution in thick biological samples, such as the mouse oocyte. We therefore developed an image processing workflow that allows improving the lateral and axial resolution of a standard confocal microscope. Our workflow comprises refractive index matching, the optimization of microscope hardware parameters and image restoration by deconvolution. We compare two different deconvolution algorithms, evaluate the necessity of denoising and establish the optimal image restoration procedure. We validate our workflow by imaging sub resolution fluorescent beads and measuring the maximum lateral and axial resolution of the confocal system. Subsequently, we apply the parameters to the imaging and data restoration of fluorescently labelled meiotic spindles of mouse oocytes. We measure a resolution increase of approximately 2-fold in the lateral and 3-fold in the axial direction throughout a depth of 60μm. This demonstrates that with our optimized workflow we reach a resolution that is comparable to 3D-SIM-imaging, but with better depth penetration for confocal images of beads and the biological sample.
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http://dx.doi.org/10.1016/j.ymeth.2016.11.003DOI Listing
February 2017

How oocytes try to get it right: spindle checkpoint control in meiosis.

Chromosoma 2016 Jun 11;125(2):321-35. Epub 2015 Aug 11.

Institut de Biologie Paris Seine (IBPS), UMR7622, Sorbonne Universités, UPMC Univ Paris 06, Paris, France.

The generation of a viable, diploid organism depends on the formation of haploid gametes, oocytes, and spermatocytes, with the correct number of chromosomes. Halving the genome requires the execution of two consecutive specialized cell divisions named meiosis I and II. Unfortunately, and in contrast to male meiosis, chromosome segregation in oocytes is error prone, with human oocytes being extraordinarily "meiotically challenged". Aneuploid oocytes, that are with the wrong number of chromosomes, give rise to aneuploid embryos when fertilized. In humans, most aneuploidies are lethal and result in spontaneous abortions. However, some trisomies survive to birth or even adulthood, such as the well-known trisomy 21, which gives rise to Down syndrome (Nagaoka et al. in Nat Rev Genet 13:493-504, 2012). A staggering 20-25 % of oocytes ready to be fertilized are aneuploid in humans. If this were not bad enough, there is an additional increase in meiotic missegregations as women get closer to menopause. A woman above 40 has a risk of more than 30 % of getting pregnant with a trisomic child. Worse still, in industrialized western societies, child birth is delayed, with women getting their first child later in life than ever. This trend has led to an increase of trisomic pregnancies by 70 % in the last 30 years (Nagaoka et al. in Nat Rev Genet 13:493-504, 2012; Schmidt et al. in Hum Reprod Update 18:29-43, 2012). To understand why errors occur so frequently during the meiotic divisions in oocytes, we review here the molecular mechanisms at works to control chromosome segregation during meiosis. An important mitotic control mechanism, namely the spindle assembly checkpoint or SAC, has been adapted to the special requirements of the meiotic divisions, and this review will focus on our current knowledge of SAC control in mammalian oocytes. Knowledge on how chromosome segregation is controlled in mammalian oocytes may help to identify risk factors important for questions related to human reproductive health.
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http://dx.doi.org/10.1007/s00412-015-0536-7DOI Listing
June 2016

Mouse oocytes depend on BubR1 for proper chromosome segregation but not for prophase I arrest.

Nat Commun 2015 Apr 21;6:6946. Epub 2015 Apr 21.

Sorbonne Universités, UPMC Université Paris 06, Institut de Biologie Paris Seine (IBPS), UMR7622, 75005 Paris, France.

Mammalian female meiosis is error prone, with rates of meiotic chromosome missegregations strongly increasing towards the end of the reproductive lifespan. A strong reduction of BubR1 has been observed in oocytes of women approaching menopause and in ovaries of aged mice, which led to the hypothesis that a gradual decline of BubR1 contributes to age-related aneuploidization. Here we employ a conditional knockout approach in mouse oocytes to dissect the meiotic roles of BubR1. We show that BubR1 is required for diverse meiotic functions, including persistent spindle assembly checkpoint activity, timing of meiosis I and the establishment of robust kinetochore-microtubule attachments in a meiosis-specific manner, but not prophase I arrest. These data reveal that BubR1 plays a multifaceted role in chromosome segregation during the first meiotic division and suggest that age-related decline of BubR1 is a key determinant of the formation of aneuploid oocytes as women approach menopause.
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http://dx.doi.org/10.1038/ncomms7946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4439927PMC
April 2015

New insights into the role of BubR1 in mitosis and beyond.

Int Rev Cell Mol Biol 2013 ;306:223-73

Institut Jacques Monod, UMR 7592 CNRS, Université Paris Diderot-Paris 7, Paris, France. Electronic address:

BubR1 is a critical component of the spindle assembly checkpoint, the surveillance mechanism that helps maintain the high fidelity of mitotic chromosome segregation by preventing cells from initiating anaphase if one or more kinetochores are not attached to the spindle. BubR1 also helps promote the establishment of stable kinetochore-microtubule attachments during prometaphase. In this chapter, we review the structure, functions, and regulation of BubR1 in these "classical roles" at the kinetochore. We discuss its recruitment to kinetochores, its assembly into the inhibitor of anaphase progression, and the importance of its posttranslational modifications. We also consider the evidence for its participation in other roles beyond mitosis, such as the meiosis-specific processes of recombination and prophase arrest of the first meiotic division, the cellular response to DNA damage, and in the regulation of centrosome and basal body function. Finally, studies are presented linking BubR1 dysfunction or misregulation to aging and human disease, particularly cancer.
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http://dx.doi.org/10.1016/B978-0-12-407694-5.00006-7DOI Listing
October 2014

Sister chromatid segregation in meiosis II: deprotection through phosphorylation.

Authors:
Katja Wassmann

Cell Cycle 2013 May 10;12(9):1352-9. Epub 2013 Apr 10.

UPMC University of Paris 6, Paris, France.

Meiotic divisions (meiosis I and II) are specialized cell divisions to generate haploid gametes. The first meiotic division with the separation of chromosomes is named reductional division. The second division, which takes place immediately after meiosis I without intervening S-phase, is equational, with the separation of sister chromatids, similar to mitosis. This meiotic segregation pattern requires the two-step removal of the cohesin complex holding sister chromatids together: cohesin is removed from chromosome arms that have been subjected to homologous recombination in meiosis I and from the centromere region in meiosis II. Cohesin in the centromere region is protected from removal in meiosis I, but this protection has to be removed--deprotected--for sister chromatid segregation in meiosis II. Whereas the mechanisms of cohesin protection are quite well understood, the mechanisms of deprotection have been largely unknown until recently. In this review I summarize our current knowledge on cohesin deprotection.
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http://dx.doi.org/10.4161/cc.24600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3674063PMC
May 2013

The PP2A inhibitor I2PP2A is essential for sister chromatid segregation in oocyte meiosis II.

Curr Biol 2013 Mar 21;23(6):485-90. Epub 2013 Feb 21.

UPMC University of Paris 6, 75005 Paris, France.

Haploid gametes are generated through two consecutive meiotic divisions, with the segregation of chromosome pairs in meiosis I and sister chromatids in meiosis II. Separase-mediated stepwise removal of cohesion, first from chromosome arms and later from the centromere region, is a prerequisite for maintaining sister chromatids together until their separation in meiosis II [1]. In all model organisms, centromeric cohesin is protected from separase-dependent removal in meiosis I through the activity of PP2A-B56 phosphatase, which is recruited to centromeres by shugoshin/MEI-S332 (Sgo) [2-5]. How this protection of centromeric cohesin is removed in meiosis II is not entirely clear; we find that all the PP2A subunits remain colocalized with the cohesin subunit Rec8 at the centromere of metaphase II chromosomes. Here, we show that sister chromatid separation in oocytes depends on a PP2A inhibitor, namely I2PP2A. I2PP2A colocalizes with the PP2A enzyme at centromeres at metaphase II, independently of bipolar attachment. When I2PP2A is depleted, sister chromatids fail to segregate during meiosis II. Our findings demonstrate that in oocytes I2PP2A is essential for faithful sister chromatid segregation by mediating deprotection of centromeric cohesin in meiosis II.
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http://dx.doi.org/10.1016/j.cub.2013.02.004DOI Listing
March 2013

Chromosome spreads with centromere staining in mouse oocytes.

Methods Mol Biol 2013 ;957:203-12

CNRS UMR7622 Biologie du Développement and UPMC Paris 6, Paris, France.

This chapter describes a technique for performing chromosome spreads from mouse oocytes. It is based on a previously described protocol (Hodges and Hunt, Chromosoma 111: 165-169, 2002), which we have modified. Chromosomes are stained with either Propidium Iodide or Hoechst. This spreading technique allows for simultaneous immunostaining of proteins associated with chromosomes. It is very useful to stain spreads with CREST serum which labels kinetochores, to be able to distinguish bivalents (chromosome pairs), dyads or univalents (paired sister chromatids), and single sister chromatids without ambiguity.
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http://dx.doi.org/10.1007/978-1-62703-191-2_14DOI Listing
April 2013

Cyclin A2 is required for sister chromatid segregation, but not separase control, in mouse oocyte meiosis.

Cell Rep 2012 Nov 1;2(5):1077-87. Epub 2012 Nov 1.

UPMC Université Paris 06, UMR7622 Laboratoire de Biologie du Développement, 9 quai St. Bernard, Paris 75005, France.

In meiosis, two specialized cell divisions allow the separation of paired chromosomes first, then of sister chromatids. Separase removes the cohesin complex holding sister chromatids together in a stepwise manner from chromosome arms in meiosis I, then from the centromere region in meiosis II. Using mouse oocytes, our study reveals that cyclin A2 promotes entry into meiosis, as well as an additional unexpected role; namely, its requirement for separase-dependent sister chromatid separation in meiosis II. Untimely cyclin A2-associated kinase activity in meiosis I leads to precocious sister separation, whereas inhibition of cyclin A2 in meiosis II prevents it. Accordingly, endogenous cyclin A is localized to kinetochores throughout meiosis II, but not in anaphase I. Additionally, we found that cyclin B1, but not cyclin A2, inhibits separase in meiosis I. These findings indicate that separase-dependent cohesin removal is differentially regulated by cyclin B1 and A2 in mammalian meiosis.
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http://dx.doi.org/10.1016/j.celrep.2012.10.002DOI Listing
November 2012

OSD1 promotes meiotic progression via APC/C inhibition and forms a regulatory network with TDM and CYCA1;2/TAM.

PLoS Genet 2012 26;8(7):e1002865. Epub 2012 Jul 26.

INRA, UMR1318, Institut Jean-Pierre Bourgin, Versailles, France.

Cell cycle control is modified at meiosis compared to mitosis, because two divisions follow a single DNA replication event. Cyclin-dependent kinases (CDKs) promote progression through both meiosis and mitosis, and a central regulator of their activity is the APC/C (Anaphase Promoting Complex/Cyclosome) that is especially required for exit from mitosis. We have shown previously that OSD1 is involved in entry into both meiosis I and meiosis II in Arabidopsis thaliana; however, the molecular mechanism by which OSD1 controls these transitions has remained unclear. Here we show that OSD1 promotes meiotic progression through APC/C inhibition. Next, we explored the functional relationships between OSD1 and the genes known to control meiotic cell cycle transitions in Arabidopsis. Like osd1, cyca1;2/tam mutation leads to a premature exit from meiosis after the first division, while tdm mutants perform an aberrant third meiotic division after normal meiosis I and II. Remarkably, while tdm is epistatic to tam, osd1 is epistatic to tdm. We further show that the expression of a non-destructible CYCA1;2/TAM provokes, like tdm, the entry into a third meiotic division. Finally, we show that CYCA1;2/TAM forms an active complex with CDKA;1 that can phosphorylate OSD1 in vitro. We thus propose that a functional network composed of OSD1, CYCA1;2/TAM, and TDM controls three key steps of meiotic progression, in which OSD1 is a meiotic APC/C inhibitor.
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http://dx.doi.org/10.1371/journal.pgen.1002865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3406007PMC
January 2013

Mps1 at kinetochores is essential for female mouse meiosis I.

Development 2011 Jun;138(11):2261-71

UPMC, 9 quai St Bernard, 75005 Paris, France.

In female meiosis, chromosome missegregations lead to the generation of aneuploid oocytes and can cause the development of trisomies or infertility. Because mammalian female meiosis I is error prone, the full functionality of control mechanisms, such as the spindle assembly checkpoint (SAC), has been put into question. The SAC monitors the correct orientation, microtubule occupancy and tension on proteinaceous structures named kinetochores. Although it has been shown previously that the SAC exists in meiosis I, where attachments are monopolar, the role of microtubule occupancy for silencing the SAC and the importance of certain essential SAC components, such as the kinase Mps1, are unknown in mammalian oocytes. Using a conditional loss-of-function approach, we address the role of Mps1 in meiotic progression and checkpoint control in meiosis I. Our data demonstrate that kinetochore localization of Mps1 is required for the proper timing of prometaphase and is essential for SAC control, chromosome alignment and aurora C localization in meiosis I. The absence of Mps1 from kinetochores severely impairs chromosome segregation in oocyte meiosis I and, therefore, fertility in mice. In addition, we settle a long-standing question in showing that kinetochore-microtubule attachments are present in prometaphase I at a time when most of the SAC protein Mad2 disappears from kinetochores.
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http://dx.doi.org/10.1242/dev.061317DOI Listing
June 2011

Meiotic homologue alignment and its quality surveillance are controlled by mouse HORMAD1.

Nat Cell Biol 2011 May 10;13(5):599-610. Epub 2011 Apr 10.

Institute of Physiological Chemistry, Technische Universität Dresden, Fiedlerstrasse 42, 01307 Dresden, Germany.

Meiotic crossover formation between homologous chromosomes (homologues) entails DNA double-strand break (DSB) formation, homology search using DSB ends, and synaptonemal-complex formation coupled with DSB repair. Meiotic progression must be prevented until DSB repair and homologue alignment are completed, to avoid the formation of aneuploid gametes. Here we show that mouse HORMAD1 ensures that sufficient numbers of processed DSBs are available for successful homology search. HORMAD1 is needed for normal synaptonemal-complex formation and for the efficient recruitment of ATR checkpoint kinase activity to unsynapsed chromatin. The latter phenomenon was proposed to be important in meiotic prophase checkpoints in both sexes. Consistent with this hypothesis, HORMAD1 is essential for the elimination of synaptonemal-complex-defective oocytes. Synaptonemal-complex formation results in HORMAD1 depletion from chromosome axes. Thus, we propose that the synaptonemal complex and HORMAD1 are key components of a negative feedback loop that coordinates meiotic progression with homologue alignment: HORMAD1 promotes homologue alignment and synaptonemal-complex formation, and synaptonemal complexes downregulate HORMAD1 function, thereby permitting progression past meiotic prophase checkpoints.
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http://dx.doi.org/10.1038/ncb2213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3087846PMC
May 2011

Phosphorylation of the spindle checkpoint protein Mad2 regulates its conformational transition.

Proc Natl Acad Sci U S A 2010 Nov 1;107(46):19772-7. Epub 2010 Nov 1.

Department of Pharmacology, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390-9041, USA.

Regulated conformational changes of proteins are critical for cellular signal transduction. The spindle checkpoint protein Mad2 is an unusual protein with two native folds: the latent open conformer (O-Mad2) and the activated closed conformer (C-Mad2). During mitosis, cytosolic O-Mad2 binds to the Mad1-Mad2 core complex at unattached kinetochores and undergoes conformational activation to become C-Mad2. C-Mad2 binds to and inhibits Cdc20, an activator of APC/C, to prevent precocious anaphase onset. Here, we show that the conformational transition of Mad2 is regulated by phosphorylation of S195 in its C-terminal region. The phospho-mimicking Mad2(S195D) mutant and the phospho-S195 Mad2 protein obtained using intein-mediated semisynthesis do not form C-Mad2 on their own. Mad2(S195D) fails to bind to Cdc20, a low-affinity ligand, but still binds to high-affinity ligands, such as Mad1 and MBP1, forming ligand-bound C-Mad2. Overexpression of Mad2(S195D) in human cells causes checkpoint defects. Our results indicate that Mad2 phosphorylation inhibits its function through differentially regulating its binding to Mad1 and Cdc20 and establish that the conformational change of Mad2 is regulated by posttranslational mechanisms.
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http://dx.doi.org/10.1073/pnas.1009000107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2993392PMC
November 2010

[Meiotic weakness: the first division].

Med Sci (Paris) 2008 Feb;24(2):197-203

Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, Box 97, 1275 York Avenue, New York 10021 , NY, USA.

Cell division is probably the most dramatic event in the life of a cell : the entire genetic material has to be equally distributed into the two daughter cells. Segregation errors have severe consequences and lead to either cell death or the generation of aneuploid cells and may cause the formation of tumors or tumor promoting mutations in somatic cells. In meiosis, they provoke the generation of aneuploid embryos and/or spontaneous abortions. Trisomies in humans, such as trisomy 21, are due to the missegregation of one chromosome in the first meiotic division in the oocyte. This review deals with the molecular mechanisms regulating the two meiotic divisions required for the generation of female haploid germ cells. Here we focus mainly on spindle assembly, and cell cycle regulation especially during the first meiotic division in mouse oocytes (excellent reviews have been written on the peculiar aspects of cell cycle regulation in meiosis II, such as the CSF arrest).
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http://dx.doi.org/10.1051/medsci/2008242197DOI Listing
February 2008

Changing Mad2 levels affects chromosome segregation and spindle assembly checkpoint control in female mouse meiosis I.

PLoS One 2007 Nov 28;2(11):e1165. Epub 2007 Nov 28.

CNRS UMR7622 Biologie du Développement, Paris, France.

The spindle assembly checkpoint (SAC) ensures correct separation of sister chromatids in somatic cells and provokes a cell cycle arrest in metaphase if one chromatid is not correctly attached to the bipolar spindle. Prolonged metaphase arrest due to overexpression of Mad2 has been shown to be deleterious to the ensuing anaphase, leading to the generation of aneuploidies and tumorigenesis. Additionally, some SAC components are essential for correct timing of prometaphase. In meiosis, we and others have shown previously that the Mad2-dependent SAC is functional during the first meiotic division in mouse oocytes. Expression of a dominant-negative form of Mad2 interferes with the SAC in metaphase I, and a knock-down approach using RNA interference accelerates anaphase onset in meiosis I. To prove unambigiously the importance of SAC control for mammalian female meiosis I we analyzed oocyte maturation in Mad2 heterozygote mice, and in oocytes overexpressing a GFP-tagged version of Mad2. In this study we show for the first time that loss of one Mad2 allele, as well as overexpression of Mad2 lead to chromosome missegregation events in meiosis I, and therefore the generation of aneuploid metaphase II oocytes. Furthermore, SAC control is impaired in mad2+/- oocytes, also leading to the generation of aneuploidies in meiosis I.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001165PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2082075PMC
November 2007

Resolution of chiasmata in oocytes requires separase-mediated proteolysis.

Cell 2006 Jul;126(1):135-46

Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria.

In yeast, resolution of chiasmata in meiosis I requires proteolytic cleavage along chromosome arms of cohesin's Rec8 subunit by separase. Since activation of separase by the anaphase-promoting complex (APC/C) is supposedly not required for meiosis I in Xenopus oocytes, it has been suggested that animal cells might resolve chiasmata by a separase-independent mechanism related to the so-called "prophase pathway" that removes cohesin from chromosome arms during mitosis. By expressing Cre recombinase from a zona pellucida promoter, we have deleted a floxed allele of separase specifically in mouse oocytes. This prevents removal of Rec8 from chromosome arms and resolution of chiasmata. It also hinders extrusion of the first polar body (PBE) and causes female sterility. mRNA encoding wild-type but not catalytically inactive separase restores chiasma resolution. Both types of mRNA restore PBE. Proteolytic activity of separase is therefore essential for Rec8's removal from chromosome arms and for chiasma resolution but not for PBE.
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http://dx.doi.org/10.1016/j.cell.2006.05.033DOI Listing
July 2006

The Mad2 spindle checkpoint protein has two distinct natively folded states.

Nat Struct Mol Biol 2004 Apr 14;11(4):338-45. Epub 2004 Mar 14.

Department of Pharmacology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.

The spindle checkpoint delays chromosome segregation in response to misaligned sister chromatids during mitosis, thus ensuring the fidelity of chromosome inheritance. Through binding to Cdc20, the Mad2 spindle checkpoint protein inhibits the target of this checkpoint, the ubiquitin protein ligase APC/C(Cdc20). We now show that without cofactor binding or covalent modification Mad2 adopts two distinct folded conformations at equilibrium (termed N1-Mad2 and N2-Mad2). The structure of N2-Mad2 has been determined by NMR spectroscopy. N2-Mad2 is much more potent in APC/C inhibition. Overexpression of a Mad2 mutant that specifically sequesters N2-Mad2 partially blocks checkpoint signaling in living cells. The two Mad2 conformers interconvert slowly in vitro, but interconversion is accelerated by a fragment of Mad1, an upstream regulator of Mad2. Our results suggest that the unusual two-state behavior of Mad2 is critical for spindle checkpoint signaling.
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http://dx.doi.org/10.1038/nsmb748DOI Listing
April 2004

The meiosis I-to-meiosis II transition in mouse oocytes requires separase activity.

Curr Biol 2003 Oct;13(20):1797-802

Division Méiotiques, Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, 75252 Paris, cedex 05, France.

Faithful segregation of homologous chromosomes during the first meiotic division is essential for further embryo development. The question at issue is whether the same mechanisms ensuring correct separation of sister chromatids in mitosis are at work during the first meiotic division. In mitosis, sister chromatids are linked by a cohesin complex holding them together until their disjunction at anaphase. Their disjunction is mediated by Separase, which cleaves the cohesin. The activation of Separase requires prior degradation of its associated inhibitor, called securin. Securin is a target of the APC/C (Anaphase Promoting Complex/Cyclosome), a cell cycle-regulated ubiquitin ligase that ubiquitinates securin at the metaphase-to-anaphase transition and thereby targets it for degradation by the 26S proteasome. After securin degradation, Separase cleaves the cohesins and triggers chromatid separation, a prerequisite for anaphase. In yeast and worms, the segregation of homologous chromosomes in meiosis I depends on the APC/C and Separase activity. Yet, it is unclear if Separase is required for the first meiotic division in vertebrates because APC/C activity is thought to be dispensable in frog oocytes. We therefore investigated if Separase activity is required for correct chromosome segregation in meiosis I in mouse oocytes.
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http://dx.doi.org/10.1016/j.cub.2003.09.032DOI Listing
October 2003

Metaphase I arrest upon activation of the Mad2-dependent spindle checkpoint in mouse oocytes.

Curr Biol 2003 Sep;13(18):1596-608

Laboratoire de Biologie Cellulaire du Développement, UMR7622, CNRS, Université Pierre et Marie Curie, 9 quai Saint Bernard, 75005 Paris, France.

Background: The importance of mitotic spindle checkpoint control has been well established during somatic cell divisions. The metaphase-to-anaphase transition takes place only when all sister chromatids have been properly attached to the bipolar spindle and are aligned at the metaphase plate. Failure of this checkpoint may lead to unequal separation of sister chromatids. On the contrary, the existence of such a checkpoint during the first meiotic division in mammalian oocytes when homologous chromosomes are segregated has remained controversial.

Results: Here, we show that mouse oocytes respond to spindle damage by a transient and reversible cell cycle arrest in metaphase I with high Maturation Promoting Factor (MPF) activity. Furthermore, the mitotic checkpoint protein Mad2 is present throughout meiotic maturation and is recruited to unattached kinetochores. Overexpression of Mad2 in meiosis I leads to a cell cycle arrest in metaphase I. Expression of a dominant-negative Mad2 protein interferes with proper spindle checkpoint arrest.

Conclusions: Errors in meiosis I cause missegregation of chromosomes and can result in the generation of aneuploid embryos with severe birth defects. In human oocytes, failures in spindle checkpoint control may be responsible for the generation of trisomies (e.g., Down Syndrome) due to chromosome missegregation in meiosis I. Up to now, the mechanisms ensuring correct separation of chromosomes in meiosis I remained unknown. Our study shows for the first time that a functional Mad2-dependent spindle checkpoint exists during the first meiotic division in mammalian oocytes.
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http://dx.doi.org/10.1016/j.cub.2003.08.052DOI Listing
September 2003

C-myc, not HER-2/neu, can predict recurrence and mortality of patients with node-negative breast cancer.

Breast Cancer Res 2003 13;5(2):R30-6. Epub 2003 Jan 13.

Frauenklinik Klinikum Ibbenbueren, Ibbenbueren, Germany.

Background: At present, node-negative, high-risk breast cancer patients cannot be identified with sufficient accuracy. Consequently, further strong prognostic factors are needed.

Methods: Among 181 node-negative breast cancer (NNBC) patients, c-myc and HER-2/neu oncogenes were identified prospectively using double differential PCR. The possible impact of amplification of those oncogenes on disease-free survival (DFS) and overall survival was examined. Furthermore, the possible effects of adjuvant therapies on rate of recurrence and mortality in oncogene-amplified NNBC patients were investigated.

Results: The prevalence rates for amplification of c-myc and HER-2/neu were 21.5% and 30.4%, respectively. On univariate analysis, c-myc-amplified NNBCs were associated with significantly shorter DFS at 36 months after the initial diagnosis (85.3% versus 97.3%). As compared with nonamplified cancers, HER-2/neu-amplified NNBCs did not exhibit any significant differences after 36 months and 95 months. Multivariate analysis indicated that c-myc amplification and tumour size, in contrast to HER-2/neu amplification, oestrogen receptor status, grading and age, were the only independent parameters for DFS. During the period of observation, we found no evidence for an impact of amplification of the oncogenes on overall survival in all cases. With respect to various adjuvant systemic therapies such as chemotherapy (cyclophosphamide, methotrexate, 5-fluorouracil; fluorouracil, epirubicin, cyclophosphamide) and endocrine therapy (tamoxifen), no significant differences were identified in oncogene-amplified NNBC patients in terms of DFS and overall survival. However, those c-myc-amplified NNBC patients who did not receive adjuvant systemic therapy exhibited significantly shorter DFS and overall survival as compared with c-myc-nonamplified patients.

Conclusion: C-myc amplification appears to be a strong prognostic marker with which to predict early recurrence in NNBC patients. C-myc-amplified NNBC patients without adjuvant systemic therapy experienced shorter DFS and overall survival.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC154146PMC
http://dx.doi.org/10.1186/bcr568DOI Listing
May 2003

Mad2 phosphorylation regulates its association with Mad1 and the APC/C.

EMBO J 2003 Feb;22(4):797-806

Cell Biology Program, Memorial Sloan-Kettering Cancer Center, Box 241, 1275 York Avenue, New York, NY 10021, USA.

Improper attachment of the mitotic spindle to the kinetochores of paired sister chromatids in mitosis is monitored by a checkpoint that leads to an arrest in early metaphase. This arrest requires the inhibitory association of Mad2 with the anaphase promoting complex/cyclosome (APC/C). It is not known how the association of Mad2 with the kinetochore and the APC/C is regulated in mitosis. Here, we demonstrate that human Mad2 is modified through phosphorylation on multiple serine residues in vivo in a cell cycle dependent manner and that only unphosphorylated Mad2 interacts with Mad1 or the APC/C in vivo. A Mad2 mutant containing serine to aspartic acid mutations mimicking the C-terminal phosphorylation events fails to interact with Mad1 or the APC/C and acts as a dominant-negative antagonist of wild-type Mad2. These data suggest that the phosphorylation state of Mad2 regulates its checkpoint activity by modulating its association with Mad1 and the APC/C.
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http://dx.doi.org/10.1093/emboj/cdg071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC145433PMC
February 2003