RESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Cromosomas/genética , Endopeptidasas/fisiología , Meiosis/genética , Proteínas Nucleares/genética , Oocitos/metabolismo , Péptido Hidrolasas/genética , Fosfoproteínas/genética , Animales , Proteínas de Ciclo Celular/genética , Células Cultivadas , Segregación Cromosómica/genética , Citocinesis/genética , Regulación hacia Abajo/genética , Endopeptidasas/genética , Femenino , Eliminación de Gen , Genes cdc/fisiología , Humanos , Masculino , Metafase/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Oocitos/citología , ARN Mensajero/genética , ARN Mensajero/metabolismo , SeparasaRESUMEN
Separase is a protease whose liberation from its inhibitory chaperone Securin triggers sister chromatid disjunction at anaphase onset in yeast by cleaving cohesin's kleisin subunit. We have created conditional knockout alleles of the mouse Separase and Securin genes. Deletion of both copies of Separase but not Securin causes embryonic lethality. Loss of Securin reduces Separase activity because deletion of just one copy of the Separase gene is lethal to embryos lacking Securin. In embryonic fibroblasts, Separase depletion blocks sister chromatid separation but does not prevent other aspects of mitosis, cytokinesis, or chromosome replication. Thus, fibroblasts lacking Separase become highly polyploid. Hepatocytes stimulated to proliferate in vivo by hepatectomy also become unusually large and polyploid in the absence of Separase but are able to regenerate functional livers. Separase depletion in bone marrow causes aplasia and the presumed death of hematopoietic cells other than erythrocytes. Destruction of sister chromatid cohesion by Separase may be a universal feature of mitosis in eukaryotic cells.
Asunto(s)
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Segregación Cromosómica/genética , Momento de Replicación del ADN/genética , Endopeptidasas/genética , Mitosis/genética , Anafase/genética , Animales , Proteínas Portadoras/genética , Línea Celular , Proteínas Cromosómicas no Histona/genética , Desarrollo Embrionario/genética , Femenino , Fibroblastos , Genes Letales/genética , Células Madre Hematopoyéticas/metabolismo , Hepatocitos , Regeneración Hepática/genética , Masculino , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Poliploidía , Securina , Separasa , CohesinasRESUMEN
Key to faithful genetic inheritance is the cohesion between sister centromeres that physically links replicated sister chromatids and is then abruptly lost at the onset of anaphase. Misregulated cohesion causes aneuploidy, birth defects and perhaps initiates cancers. Loss of centromere cohesion is controlled by the spindle checkpoint and is thought to depend on a ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC). But here we present evidence that the APC pathway is dispensable for centromere separation at anaphase in mammals, and that anaphase proceeds in the presence of cyclin B and securin. Arm separation is perturbed in the absence of APC, compromising the fidelity of segregation, but full sister chromatid separation is achieved after a delayed anaphase. Thereafter, cells arrest terminally in telophase with high levels of cyclin B. Extending these findings we provide evidence that the spindle checkpoint regulates centromere cohesion through an APC-independent pathway. We propose that this Centromere Linkage Pathway (CLiP) is a second branch that stems from the spindle checkpoint to regulate cohesion preferentially at the centromeres and that Sgo1 is one of its components.
Asunto(s)
Anafase/fisiología , Centrómero/fisiología , Intercambio de Cromátides Hermanas/fisiología , Huso Acromático/fisiología , Complejos de Ubiquitina-Proteína Ligasa/fisiología , Anafase/genética , Ciclosoma-Complejo Promotor de la Anafase , Animales , Células Cultivadas , Centrómero/química , Centrómero/genética , Células HeLa , Humanos , Ratones , ARN Interferente Pequeño/genética , Intercambio de Cromátides Hermanas/genética , Huso Acromático/química , Huso Acromático/genética , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/genéticaRESUMEN
Loss of centromere cohesion during anaphase in human cells is regulated by the spindle assembly checkpoint and is thought to depend on a ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC). APC-Cdc20 adds ubiquitin chains to securin inducing its destruction by the proteasome and these events correlate with the loss of sister chromatid cohesion and the onset of anaphase. But whether securin destruction is necessary and sufficient for anaphase initiation is not clear. Therefore, we asked if proteasome activity is needed for anaphase onset in human cells that lack securin. We find that even in the absence of securin, a metaphase block with cohered sister centromeres can be enforced in the absence of proteasome activity. Therefore, other targets of the proteasome must be degraded to allow anaphase onset.
Asunto(s)
Centrómero/enzimología , Proteínas de Neoplasias/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/fisiología , Anafase/fisiología , Línea Celular Tumoral , Centrómero/fisiología , Segregación Cromosómica/fisiología , Humanos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Complejo de la Endopetidasa Proteasomal/metabolismo , SecurinaRESUMEN
The anaphase-promoting complex or cyclosome (APC/C) is an ubiquitin protein ligase that together with Cdc20 and Cdh1 targets mitotic proteins for degradation by the proteosome. APC-Cdc20 activity during mitosis triggers anaphase by destroying securin and cyclins. APC-Cdh1 promotes degradation of cyclins and other proteins during G(1). We show that loss of APC/C during embryogenesis is early lethal before embryonic day E6.5 (E6.5). To investigate the role of APC/C in quiescent cells, we conditionally inactivated the subunit Apc2 in mice. Deletion of Apc2 in quiescent hepatocytes caused re-entry into the cell cycle and arrest in metaphase, resulting in liver failure. Re-entry into the cell cycle either occurred without any proliferative stimulus or could be easily induced. We demonstrate that the APC has an additional function to prevent hepatocytes from unscheduled re-entry into the cell cycle.
