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1.
EMBO J ; 40(7): e106797, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33644892

RESUMEN

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.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cinetocoros/metabolismo , Meiosis , Animales , Células Cultivadas , Cromátides/genética , Cromátides/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Oocitos/metabolismo , Cohesinas
3.
J Cell Sci ; 126(Pt 17): 3916-26, 2013 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-23781026

RESUMEN

Entry into mitosis or meiosis relies on the coordinated action of kinases and phosphatases that ultimately leads to the activation of Cyclin-B-Cdk1, also known as MPF for M-phase promoting factor. Vertebrate oocytes are blocked in prophase of the first meiotic division, an arrest that is tightly controlled by high PKA activity. Re-entry into meiosis depends on activation of Cdk1, which obeys a two-step mechanism: a catalytic amount of Cdk1 is generated in a PKA and protein-synthesis-dependent manner; then a regulatory network known as the MPF auto-amplification loop is initiated. This second step is independent of PKA and protein synthesis. However, none of the molecular components of the auto-amplification loop identified so far act independently of PKA. Therefore, the protein rendering this process independent of PKA in oocytes remains unknown. Using a physiologically intact cell system, the Xenopus oocyte, we show that the phosphorylation of ARPP19 at S67 by the Greatwall kinase promotes its binding to the PP2A-B55δ phosphatase, thus inhibiting its activity. This process is controlled by Cdk1 and has an essential role within the Cdk1 auto-amplification loop for entry into the first meiotic division. Moreover, once phosphorylated by Greatwall, ARPP19 escapes the negative regulation exerted by PKA. It also promotes activation of MPF independently of protein synthesis, provided that a small amount of Mos is present. Taken together, these findings reveal that PP2A-B55δ, Greatwall and ARPP19 are not only required for entry into meiotic divisions, but are also pivotal effectors within the Cdk1 auto-regulatory loop responsible for its independence with respect to the PKA-negative control.


Asunto(s)
Factor Promotor de Maduración/metabolismo , Oocitos/metabolismo , Fosfoproteínas/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Regulación hacia Abajo , Femenino , Factor Promotor de Maduración/genética , Meiosis/fisiología , Fosforilación , Xenopus laevis/metabolismo
4.
Development ; 138(11): 2261-71, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21558374

RESUMEN

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.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Meiosis/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Segregación Cromosómica , Femenino , Técnica del Anticuerpo Fluorescente , Proteínas Mad2 , Ratones , Ratones Transgénicos , Microtúbulos/metabolismo , Oocitos/citología , Oocitos/metabolismo , Reacción en Cadena de la Polimerasa , Huso Acromático/metabolismo
5.
Nat Cell Biol ; 9(5): 565-72, 2007 May.
Artículo en Inglés | MEDLINE | ID: mdl-17417628

RESUMEN

Mad2 has a key role in the spindle-assembly checkpoint (SAC) - the mechanism delaying anaphase onset until all chromosomes correctly attach to the spindle. Here, we show that unlike every other reported case of SAC inactivation in metazoans, mad2-null Drosophila are viable and fertile, and their cells almost always divide correctly despite having no SAC and an accelerated 'clock', which is caused by premature degradation of cyclin B. Mitosis in Drosophila does not need the SAC because correct chromosome attachment is achieved very rapidly, before even the cell lacking Mad2 can initiate anaphase. Experimentally reducing spindle-assembly efficiency renders the cells Mad2-dependent. In fact, the robustness of the SAC may generally mask minor mitotic defects of mutations affecting spindle function. The reported lethality of other Drosophila SAC mutations may be explained by their multifunctionality, and thus the 'checkpoint' phenotypes previously ascribed to these mutations should be considered the consequence of eliminating both the checkpoint and a second mitotic function.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica , Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Mitosis/fisiología , Huso Acromático/metabolismo , Anafase/fisiología , Ciclosoma-Complejo Promotor de la Anafase , Animales , Proteínas de Ciclo Celular/genética , Colchicina/farmacología , Ciclina B/metabolismo , Drosophila/efectos de los fármacos , Drosophila/embriología , Drosophila/genética , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Proteínas de Homeodominio/metabolismo , Cinetocoros/metabolismo , Larva/fisiología , Proteínas Mad2 , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis/efectos de los fármacos , Mutación , Fenotipo , Huso Acromático/efectos de los fármacos , Factores de Tiempo , Moduladores de Tubulina/farmacología , Complejos de Ubiquitina-Proteína Ligasa/metabolismo
6.
Curr Biol ; 32(10): 2281-2290.e4, 2022 05 23.
Artículo en Inglés | MEDLINE | ID: mdl-35385691

RESUMEN

To generate haploid gametes, cohesin is removed in a stepwise manner from chromosome arms in meiosis I and the centromere region in meiosis II to segregate chromosomes and sister chromatids, respectively. Meiotic cohesin removal requires cleavage of the meiosis-specific kleisin subunit Rec8 by the protease separase.1,2 In yeast and C. elegans, Rec8 on chromosome arms has to be phosphorylated to be cleaved in meiosis I,3-7 whereas Rec8 at the centromere is protected from cleavage by the action of PP2A-B56.8-10 However, in mammalian meiosis, it is unknown whether Rec8 has to be equally phosphorylated for cleavage, and if so, the identity of the relevant kinase(s). This is due to technical challenges, as Rec8 is poorly conserved, preventing a direct translation of the knowledge gained from model systems such as yeast and C. elegans to mammals. Additionally, there is no turnover of Rec8 after cohesion establishment, preventing phosphomutant analysis of functional Rec8. To address the very basic question of whether Rec8 cleavage requires its phosphorylation in mammals, we adapted a biosensor that detects separase activity to study Rec8 cleavage in single mouse oocytes by live imaging. Crucially, through phosphomutant analysis, we identified phosphorylation sites in Rec8 promoting cleavage. We found that Rec8 cleavage depends on Aurora B/C kinase activities and identified an aminoacid residue that is phosphorylated in vivo. Accordingly, inhibition of Aurora B/C kinases during meiotic maturation impairs endogenous Rec8 phosphorylation and chromosome segregation.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Mamíferos/genética , Meiosis , Ratones , Oocitos/metabolismo , Fosforilación , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Separasa/metabolismo
7.
Curr Biol ; 15(9): 856-61, 2005 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-15886105

RESUMEN

Compromising the activity of the spindle checkpoint permits mitotic exit in the presence of unattached kinetochores and, consequently, greatly increases the rate of aneuploidy in the daughter cells. The metazoan checkpoint mechanism is more complex than in yeast in that it requires additional proteins and activities besides the classical Mads and Bubs. Among these are Rod, Zw10, and Zwilch, components of a 700 Kdal complex (Rod/Zw10) that is required for recruitment of dynein/dynactin to kinetochores but whose role in the checkpoint is poorly understood. The dynamics of Rod and Mad2, examined in different organisms, show intriguing similarities as well as apparent differences. Here we simultaneously follow GFP-Mad2 and RFP-Rod and find they are in fact closely associated throughout early mitosis. They accumulate simultaneously on kinetochores and are shed together along microtubule fibers after attachment. Their behavior and position within attached kinetochores is distinct from that of BubR1; Mad2 and Rod colocalize to the outermost kinetochore region (the corona), whereas BubR1 is slightly more interior. Moreover, Mad2, but not BubR1, Bub1, Bub3, or Mps1, requires Rod/Zw10 for its accumulation on unattached kinetochores. Rod/Zw10 thus contributes to checkpoint activation by promoting Mad2 recruitment and to checkpoint inactivation by recruiting dynein/dynactin that subsequently removes Mad2 from attached kinetochores.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis/fisiología , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Animales , Western Blotting , Encéfalo/metabolismo , Clonación Molecular , Cartilla de ADN , Drosophila , Dineínas/metabolismo , Ganglios de Invertebrados/metabolismo , Proteínas Fluorescentes Verdes , Larva/metabolismo , Proteínas Luminiscentes , Microtúbulos/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteína Fluorescente Roja
8.
Curr Biol ; 28(1): 130-139.e3, 2018 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-29276128

RESUMEN

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.


Asunto(s)
Cinetocoros/fisiología , Puntos de Control de la Fase M del Ciclo Celular/fisiología , Meiosis/fisiología , Oocitos/fisiología , Animales , División Celular/efectos de los fármacos , División Celular/fisiología , Cisteína/análogos & derivados , Cisteína/farmacología , Femenino , Cinesinas/antagonistas & inhibidores , Cinetocoros/efectos de los fármacos , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Ratones , Oocitos/efectos de los fármacos , Paclitaxel/farmacología , Pirimidinas/farmacología , Tionas/farmacología , Moduladores de Tubulina/farmacología
9.
Nat Commun ; 8(1): 694, 2017 09 25.
Artículo en Inglés | MEDLINE | ID: mdl-28947820

RESUMEN

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.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Meiosis/fisiología , Oocitos/citología , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Proteínas de Ciclo Celular/análisis , Centrómero/ultraestructura , Ratones , Oocitos/metabolismo , Oocitos/ultraestructura , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Cohesinas
10.
Nat Commun ; 6: 6946, 2015 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-25897860

RESUMEN

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.


Asunto(s)
Puntos de Control del Ciclo Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica/fisiología , Profase Meiótica I/fisiología , Oocitos/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Femenino , Leviviridae , Ratones , Ratones Endogámicos , Proteínas Serina-Treonina Quinasas/genética
11.
PLoS One ; 5(2): e9285, 2010 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-20174573

RESUMEN

BACKGROUND: In Drosophila, each external sensory organ originates from the division of a unique precursor cell (the sensory organ precursor cell or SOP). Each SOP is specified from a cluster of equivalent cells, called a proneural cluster, all of them competent to become SOP. Although, it is well known how SOP cells are selected from proneural clusters, little is known about the downstream genes that are regulated during SOP fate specification. METHODOLOGY/PRINCIPAL FINDINGS: In order to better understand the mechanism involved in the specification of these precursor cells, we combined laser microdissection, toisolate SOP cells, with transcriptome analysis, to study their RNA profile. Using this procedure, we found that genes that exhibit a 2-fold or greater expression in SOPs versus epithelial cells were mainly associated with Gene Ontology (GO) terms related with cell fate determination and sensory organ specification. Furthermore, we found that several genes such as pebbled/hindsight, scabrous, miranda, senseless, or cut, known to be expressed in SOP cells by independent procedures, are particularly detected in laser microdissected SOP cells rather than in epithelial cells. CONCLUSIONS/SIGNIFICANCE: These results confirm the feasibility and the specificity of our laser microdissection based procedure. We anticipate that this analysis will give new insight into the selection and specification of neural precursor cells.


Asunto(s)
Drosophila/genética , Microdisección/métodos , Células Receptoras Sensoriales/metabolismo , Células Madre/metabolismo , Animales , Diferenciación Celular/genética , Drosophila/citología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Estudios de Factibilidad , Perfilación de la Expresión Génica , Rayos Láser , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Receptoras Sensoriales/citología , Células Madre/citología
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