RESUMEN
For correct chromosome segregation in mitosis, sister kinetochores must interact with microtubules from opposite spindle poles (biorientation). For this, aberrant kinetochore-microtubule interaction must be resolved (error correction) by Aurora B kinase. Once biorientation is formed, tension is applied on kinetochore-microtubule interaction, stabilizing this interaction. The mechanism for this tension-dependent process has been debated. Here, we study how Aurora B localizations at different kinetochore sites affect the biorientation establishment and maintenance in budding yeast. Without the physiological Aurora B-INCENP recruitment mechanisms, engineered recruitment of Aurora B-INCENP to the inner kinetochore, but not to the outer kinetochore, prior to biorientation supports the subsequent biorientation establishment. Moreover, when the physiological Aurora B-INCENP recruitment mechanisms are present, an engineered Aurora B-INCENP recruitment to the outer kinetochore, but not to the inner kinetochore, during metaphase (after biorientation establishment) disrupts biorientation, which is dependent on the Aurora B kinase activity. These results suggest that the spatial separation of Aurora B from its outer kinetochore substrates is required to stabilize kinetochore-microtubule interaction when biorientation is formed and tension is applied on this interaction. Meanwhile, Aurora B exhibits dynamic turnover on the centromere/kinetochore during early mitosis, a process thought to be crucial for error correction and biorientation. However, using the engineered Aurora B-INCENP recruitment to the inner kinetochore, we demonstrate that, even without such a turnover, Aurora B-INCENP can efficiently support biorientation. Our study provides important insights into how Aurora B promotes error correction for biorientation in a tension-dependent manner.
Asunto(s)
Segregación Cromosómica , Cinetocoros , Aurora Quinasa B/genética , Centrómero , Microtúbulos , MitosisRESUMEN
Accurate genome segregation in mitosis requires that all chromosomes are bioriented on the spindle. Cells monitor biorientation by sensing tension across sister centromeres. Chromosomes that are not bioriented have low centromere tension, which allows Aurora B (yeast Ipl1) to perform error correction that locally loosens kinetochore-microtubule attachments to allow detachment of microtubules and fresh attempts at achieving biorientation. However, it is not known whether low tension recruits Aurora B to centromeres or, alternatively, whether low tension directly activates Aurora B already localized at centromeres. In this work, we experimentally induced low tension in metaphase Saccharomyces cerevisiae yeast cells, then monitored Ipl1 localization. We find low tension recruits Ipl1 to centromeres. Furthermore, low tension-induced Ipl1 recruitment depended on Bub1, which is known to provide a binding site for Ipl1. In contrast, Top2, which can also recruit Ipl1 to centromeres, was not required. Our results demonstrate cells are sensitive to low tension at centromeres and respond by actively recruiting Ip1l for error correction.
Asunto(s)
Cinetocoros , Saccharomyces cerevisiae , Aurora Quinasa B/genética , Aurora Quinasa B/metabolismo , Centrómero/metabolismo , Segregación Cromosómica , Proteínas Fúngicas/metabolismo , Cinetocoros/metabolismo , Metafase , Microtúbulos/metabolismo , Mitosis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Centromere identity is specified epigenetically by specialized nucleosomes containing the evolutionarily conserved centromeric histone H3 variant (Cse4 in budding yeast, CENP-A in humans) which is essential for faithful chromosome segregation. However, the mechanisms of epigenetic regulation of Cse4 have not been clearly defined. We have identified two kinases, Cdc5 (Plk1 in humans) and Ipl1 (Aurora B kinase in humans) that phosphorylate Cse4 to prevent chromosomal instability (CIN). Cdc5 associates with Cse4 in mitosis and Cdc5-mediated phosphorylation of Cse4 is coincident with the centromeric enrichment of Cdc5 during metaphase. Defects in Cdc5-mediated Cse4 phosphorylation causes CIN, whereas constitutive association of Cdc5 with Cse4 results in lethality. Cse4 is also a substrate for Ipl1 and phospho-mimetic cse4 mutants suppress growth defects of ipl1 and Ipl1 kinetochore substrate mutants, namely dam1 spc34 and ndc80. Ipl1-mediated phosphorylation of Cse4 regulates kinetochore-microtubule interactions and chromosome biorientation. We propose that collaboration of Cdc5- and Ipl1-mediated phosphorylation of Cse4 modulates kinetochore structure and function, and chromosome biorientation. These findings demonstrate how phosphorylation of Cse4 regulates the integrity of the kinetochore, and acts as an epigenetic marker for mitotic control.
Asunto(s)
Aurora Quinasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Mitosis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Aurora Quinasas/genética , Proteínas de Ciclo Celular/genética , Centrómero/metabolismo , Proteína A Centromérica/genética , Proteína A Centromérica/metabolismo , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/genética , Proteínas de Unión al ADN/genética , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
For proper chromosome segregation in mitosis, sister kinetochores must interact with microtubules from opposite spindle poles (chromosome bi-orientation) [1, 2]. To promote bi-orientation, Aurora B kinase disrupts aberrant kinetochore-microtubule interactions [3-6]. It has long been debated how Aurora B halts this action when bi-orientation is established and tension is applied across sister kinetochores. A popular explanation for it is that, upon bi-orientation, sister kinetochores are pulled in opposite directions, stretching the outer kinetochores [7, 8] and moving Aurora B substrates away from Aurora-B-localizing sites at centromeres (spatial separation model) [3, 5, 9]. This model predicts that Aurora B localization at centromeres is required for bi-orientation. However, this notion was challenged by the observation that Bir1 (yeast survivin), which recruits Ipl1-Sli15 (yeast Aurora B-INCENP) to centromeres, can become dispensable for bi-orientation [10]. This raised the possibility that Aurora B localization at centromeres is dispensable for bi-orientation. Alternatively, there might be a Bir1-independent mechanism for recruiting Ipl1-Sli15 to centromeres or inner kinetochores [5, 9]. Here, we show that the COMA inner kinetochore sub-complex physically interacts with Sli15, recruits Ipl1-Sli15 to the inner kinetochore, and promotes chromosome bi-orientation, independently of Bir1, in budding yeast. Moreover, using an engineered recruitment of Ipl1-Sli15 to the inner kinetochore when both Bir1 and COMA are defective, we show that localization of Ipl1-Sli15 at centromeres or inner kinetochores is required for bi-orientation. Our results give important insight into how Aurora B disrupts kinetochore-microtubule interaction in a tension-dependent manner to promote chromosome bi-orientation.
Asunto(s)
Aurora Quinasas/genética , Centrómero/metabolismo , Cinetocoros/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Aurora Quinasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Proper segregation of the replicated genome requires that kinetochores form and maintain bioriented, amphitelic attachments to microtubules from opposite spindle poles and eliminate erroneous, syntelic attachments to microtubules from the same spindle pole. Phosphorylation of kinetochore proteins destabilizes low-tension kinetochore-microtubule attachments, yet tension stabilizes bioriented attachments. This conundrum for forming high-tension amphitelic attachments is recognized as the "initiation problem of biorientation (IPBO)." A delay before kinetochore-microtubule detachment solves the IPBO, but it lacks a mechanistic framework. We developed a stochastic mathematical model for kinetochore-microtubule error correction in yeast that reveals: (1) under low chromatin tension, requiring a large number of phosphorylation events at multiple sites to achieve detachment provides the necessary delay; and (2) kinetochore-induced microtubule depolymerization generates tension in amphitelic, but not syntelic, attachments. With these requirements, the model provides a mechanistic framework for the delay before detachment to solve the IPBO and demonstrates the high degree of amphitely observed experimentally for wild-type spindles under optimal conditions.
Asunto(s)
Mitosis/genética , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Polos del Huso/genética , Cromatina/genética , Segregación Cromosómica/genética , Cinetocoros/fisiología , Microtúbulos/genéticaRESUMEN
Kinetochores connect centromeric nucleosomes with mitotic-spindle microtubules through conserved, cross-interacting protein subassemblies. In budding yeast, the heterotetrameric MIND complex (Mtw1, Nnf1, Nsl1, Dsn1), ortholog of the metazoan Mis12 complex, joins the centromere-proximal components, Mif2 and COMA, with the principal microtubule-binding component, the Ndc80 complex (Ndc80C). We report the crystal structure of Kluyveromyces lactis MIND and examine its partner interactions, to understand the connection from a centromeric nucleosome to a much larger microtubule. MIND resembles an elongated, asymmetric Y; two globular heads project from a coiled-coil shaft. An N-terminal extension of Dsn1 from one head regulates interactions of the other head, blocking binding of Mif2 and COMA. Dsn1 phosphorylation by Ipl1/Aurora B relieves this autoinhibition, enabling MIND to join an assembling kinetochore. A C-terminal extension of Dsn1 recruits Ndc80C to the opposite end of the shaft. The structure and properties of MIND show how it integrates phospho-regulatory inputs for kinetochore assembly and disassembly.
Asunto(s)
Proteínas Cromosómicas no Histona/química , Proteínas Fúngicas/química , Cinetocoros/química , Kluyveromyces/química , Complejos Multiproteicos/química , Proteínas Cromosómicas no Histona/metabolismo , Cristalografía por Rayos X , Proteínas Fúngicas/metabolismo , Cinetocoros/metabolismo , Kluyveromyces/citología , Kluyveromyces/metabolismo , Complejos Multiproteicos/metabolismoRESUMEN
Aurora B kinase regulates the proper biorientation of sister chromatids during mitosis. Lack of Aurora B kinase function results in the inability to correct erroneous kinetochore-microtubule attachments and gives rise to aneuploidy. Interestingly, increased Aurora B activity also leads to problems with chromosome segregation, and overexpression of this kinase has been observed in various types of cancer. However, little is known about the mechanisms by which an increase in Aurora B kinase activity can impair mitotic progression and cell viability. Here, using a yeast model, we demonstrate that increased Aurora B activity as a result of the overexpression of the Aurora B and inner centromere protein homologs triggers defects in chromosome segregation by promoting the continuous disruption of chromosome-microtubule attachments even when sister chromatids are correctly bioriented. This disruption leads to a constitutive activation of the spindle-assembly checkpoint, which therefore causes a lack of cytokinesis even though spindle elongation and chromosome segregation take place. Finally, we demonstrate that this increase in Aurora B activity causes premature collapse of the mitotic spindle by promoting instability of the spindle midzone.
Asunto(s)
Aurora Quinasa B/metabolismo , Cromosomas Fúngicos/metabolismo , Cinetocoros/enzimología , Microtúbulos/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Huso Acromático/enzimología , Aurora Quinasa B/genética , Cromátides/enzimología , Cromátides/genética , Cromosomas Fúngicos/genética , Microtúbulos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Huso Acromático/genéticaRESUMEN
In meiosis I, homologous chromosomes pair and then attach to the spindle so that the homologs can be pulled apart at anaphase I. The segregation of homologs before pairing would be catastrophic. We describe two mechanisms that prevent this. First, in early meiosis, Ipl1, the budding yeast homolog of the mammalian Aurora B kinase, triggers shedding of a kinetochore protein, preventing microtubule attachment. Second, Ipl1 localizes to the spindle pole bodies (SPBs), where it blocks spindle assembly. These processes are reversed upon expression of Ndt80. Previous studies have shown that Ndt80 is expressed when homologs have successfully partnered, and this triggers a rise in the levels of cyclin-dependent kinase (CDK). We found that CDK phosphorylates Ipl1, delocalizing it from SPBs, triggering spindle assembly. At the same time, kinetochores reassemble. Thus, dual mechanisms controlled by Ipl1 and Ntd80 coordinate chromosome and spindle behaviors to prevent the attachment of unpartnered chromosomes to the meiotic spindle.
Asunto(s)
Aurora Quinasas/metabolismo , Segregación Cromosómica/fisiología , Meiosis/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Aurora Quinasas/genética , Segregación Cromosómica/genética , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica , Cinetocoros/metabolismo , Meiosis/genética , Microtúbulos/metabolismo , Fosforilación , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Huso Acromático/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Nuclear transport is a dynamic process that can be modulated in response to changes in cellular physiology. We recently reported that the transport activity of yeast nuclear pore complexes (NPCs) is altered in response to kinetochore-microtubule (KT-MT) interaction defects. Specifically, KT detachment from MTs activates a signaling pathway that prevents the nuclear import of cargos by the nuclear transport factor Kap121p. This loss of Kap121p-mediated import is thought to influence the nuclear environment, including the phosphorylation state of nuclear proteins. A key regulator of this process is the spindle assembly checkpoint protein Mad1p. In response to unattached KTs, Mad1p dynamically cycles between NPCs and KTs. This cycling appears to induce NPC molecular rearrangements that prevent the nuclear import of Kap121p-cargo complexes. Here, we discuss the underlying mechanisms and the physiological relevance of Mad1p cycling and the inhibition of Kap121p-mediated nuclear import, focusing on outstanding questions within the pathway.
Asunto(s)
Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Huso Acromático/metabolismo , Transporte Activo de Núcleo Celular , Animales , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
The kinetochore is the macromolecular protein complex that mediates chromosome segregation. The Dsn1 component is crucial for kinetochore assembly and is phosphorylated by the Aurora B kinase. We found that Aurora B phosphorylation of Dsn1 promotes the interaction between outer and inner kinetochore proteins in budding yeast.
Asunto(s)
Aurora Quinasa B/genética , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Aurora Quinasa B/metabolismo , Regulación Fúngica de la Expresión Génica , Cinetocoros/metabolismo , Cinetocoros/ultraestructura , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Mitosis/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Mapas de Interacción de Proteínas/genética , Saccharomyces cerevisiae/citologíaRESUMEN
Ipl1/Aurora B is the catalytic subunit of a protein kinase complex required for chromosome segregation and nuclear division. Before anaphase, Ipl1 is required to establish proper kinetochore-microtubule associations and to regulate the spindle assembly checkpoint (SAC). The phosphatase Glc7/PP1 opposes Ipl1 for these activities. To investigate Ipl1 and Glc7 regulation in more detail, we isolated and characterized mutations in the yeast Saccharomyces cerevisiae that raise the restrictive temperature of the ipl-2 mutant. These suppressors include three intragenic, second-site revertants in IPL1; 17 mutations in Glc7 phosphatase components (GLC7, SDS22, YPI1); two mutations in SHP1, encoding a regulator of the AAA ATPase Cdc48; and a mutation in TCO89, encoding a subunit of the TOR Complex 1. Two revertants contain missense mutations in microtubule binding components of the kinetochore. rev76 contains the missense mutation duo1-S115F, which alters an essential component of the DAM1/DASH complex. The mutant is cold sensitive and arrests in G2/M due to activation of the SAC. rev8 contains the missense mutation ndc80-K204E. K204 of Ndc80 corresponds to K166 of human Ndc80 and the human Ndc80 K166E variant was previously shown to be defective for microtubule binding in vitro. In a wild-type IPL1 background, ndc80-K204E cells grow slowly and the SAC is activated. The slow growth and cell cycle delay of ndc80-K204E cells are partially alleviated by the ipl1-2 mutation. These data provide biological confirmation of a biochemically based model for the effect of phosphorylation on Ndc80 function.