RESUMEN
A selective disruption of the mouse CENP-E gene was generated to test how this kinetochore-associated, kinesin-like protein contributes to chromosome segregation. The removal of CENP-E in primary cells produced spindles in which some metaphase chromosomes lay juxtaposed to a spindle pole, despite the absence of microtubules stably bound to their kinetochores. Most CENP-E-free chromosomes moved to the spindle equator, but their kinetochores bound only half the normal number of microtubules. Deletion of CENP-E in embryos led to early developmental arrest. Selective deletion of CENP-E in liver revealed that tissue regeneration after chemical damage was accompanied by aberrant mitoses marked by chromosome missegregation. CENP-E is thus essential for the maintenance of chromosomal stability through efficient stabilization of microtubule capture at kinetochores.
Asunto(s)
Proteínas Portadoras , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/fisiología , Segregación Cromosómica , Cromosomas/fisiología , Cinetocoros/ultraestructura , Microtúbulos/fisiología , Adenoviridae/genética , Animales , Proteínas de Unión al Calcio/fisiología , Tetracloruro de Carbono/toxicidad , Proteínas de Ciclo Celular , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas , Cromosomas/ultraestructura , Cruzamientos Genéticos , Fibroblastos , Proteínas Fúngicas/fisiología , Eliminación de Gen , Biblioteca Genómica , Genotipo , Hepatocitos/patología , Integrasas/metabolismo , Cinetocoros/fisiología , Hepatopatías/patología , Regeneración Hepática/genética , Regeneración Hepática/fisiología , Proteínas Mad2 , Ratones/embriología , Microtúbulos/ultraestructura , Mitosis , Proteínas Nucleares , Células Madre/citología , Células Madre/fisiología , Proteínas Virales/metabolismoRESUMEN
Centromere-associated protein-E (CENP-E) is an essential mitotic kinesin that is required for efficient, stable microtubule capture at kinetochores. It also directly binds to BubR1, a kinetochore-associated kinase implicated in the mitotic checkpoint, the major cell cycle control pathway in which unattached kinetochores prevent anaphase onset. Here, we show that single unattached kinetochores depleted of CENP-E cannot block entry into anaphase, resulting in aneuploidy in 25% of divisions in primary mouse fibroblasts in vitro and in 95% of regenerating hepatocytes in vivo. Without CENP-E, diminished levels of BubR1 are recruited to kinetochores and BubR1 kinase activity remains at basal levels. CENP-E binds to and directly stimulates the kinase activity of purified BubR1 in vitro. Thus, CENP-E is required for enhancing recruitment of its binding partner BubR1 to each unattached kinetochore and for stimulating BubR1 kinase activity, implicating it as an essential amplifier of a basal mitotic checkpoint signal.