RESUMO
The fungal-specific heterodecameric outer kinetochore DASH complex facilitates the interaction of kinetochores with spindle microtubules. In budding yeast, where kinetochores bind a single microtubule, the DASH complex is essential, and phosphorylation of Dam1 by the Aurora kinase homologue, Ipl1, causes detachment of kinetochores from spindle microtubules. We demonstrate that in the distantly related fission yeast, where the DASH complex is not essential for viability and kinetochores bind multiple microtubules, Dam1 is instead phosphorylated on serine 143 by the Polo kinase homologue, Plo1, during prometaphase and metaphase. This phosphorylation site is conserved in most fungal Dam1 proteins, including budding yeast Dam1. We show that Dam1 phosphorylation by Plo1 is dispensable for DASH assembly and chromosome retrieval but instead aids tension-dependent chromosome bi-orientation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Aurora Quinases , FosforilaçãoRESUMO
Regulated interaction between kinetochores and the mitotic spindle is essential for the fidelity of chromosome segregation. Potentially deleterious attachments are corrected during prometaphase and metaphase. Correct attachments must persist during anaphase, when spindle-generated forces separate chromosomes to opposite poles. In yeast, the heterodecameric DASH complex plays a vital pole in maintaining this link. In vitro DASH forms both oligomeric patches and rings that can form load-bearing attachments with the tips of polymerising and depolymerising microtubules. In vivo, DASH localises primarily at the kinetochore, and has a role maintaining correct attachment between spindles and chromosomes in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. Recent work has begun to describe how DASH acts alongside other components of the outer kinetochore to create a dynamic, regulated kinetochore-microtubule interface. Here, we review some of the key experiments into DASH function and discuss their implications for the nature of kinetochore-microtubule attachments in yeast and other organisms.
Assuntos
Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animais , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Leveduras/metabolismoRESUMO
The onset of anaphase is triggered by activation of the anaphase-promoting complex/cyclosome (APC/C) following silencing of the spindle assembly checkpoint (SAC). APC/C triggers ubiquitination of Securin and Cyclin B, which leads to loss of sister chromatid cohesion and inactivation of Cyclin B/Cdk1, respectively. This promotes relocalization of Aurora B kinase and other components of the chromosome passenger complex (CPC) from centromeres to the spindle midzone. In fission yeast, this is mediated by Clp1 phosphatase-dependent interaction of CPC with Klp9/MKLP2 (kinesin-6). When this interaction is disrupted, kinetochores bi-orient normally, but APC/C activation is delayed via a mechanism that requires Sgo2 and some (Bub1, Mph1/Mps1, and Mad3), but not all (Mad1 and Mad2), components of the SAC and the first, but not second, lysine, glutamic acid, glutamine (KEN) box in Mad3. These data indicate that interaction of CPC with Klp9 terminates a Sgo2-dependent, but Mad2-independent, APC/C-inhibitory pathway that is distinct from the canonical SAC.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Schizosaccharomyces/fisiologia , Anáfase , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Asparagina/metabolismo , Aurora Quinase B/metabolismo , Ciclo Celular/fisiologia , Centrômero/metabolismo , Centrômero/fisiologia , Ciclina B/metabolismo , Ácido Glutâmico/metabolismo , Cinetocoros/metabolismo , Cinetocoros/fisiologia , Lisina/metabolismo , Proteínas Nucleares/metabolismo , Fuso Acromático/metabolismo , Fuso Acromático/fisiologiaRESUMO
The spindle assembly checkpoint (SAC) is the major surveillance system that ensures that sister chromatids do not separate until all chromosomes are correctly bioriented during mitosis. Components of the checkpoint include Mad1, Mad2, Mad3 (BubR1), Bub3, and the kinases Bub1, Mph1 (Mps1), and Aurora B. Checkpoint proteins are recruited to kinetochores when individual kinetochores are not bound to spindle microtubules or not under tension. Kinetochore association of Mad2 causes it to undergo a conformational change, which promotes its association to Mad3 and Cdc20 to form the mitotic checkpoint complex (MCC). The MCC inhibits the anaphase-promoting complex/cyclosome (APC/C) until the checkpoint is satisfied. SAC silencing derepresses Cdc20-APC/C activity. This triggers the polyubiquitination of securin and cyclin, which promotes the dissolution of sister chromatid cohesion and mitotic progression. We, and others, recently showed that association of PP1 to the Spc7/Spc105/KNL1 family of kinetochore proteins is necessary to stabilize microtubule-kinetochore attachments and silence the SAC. We now report that phosphorylation of the conserved MELT motifs in Spc7 by Mph1 (Mps1) recruits Bub1 and Bub3 to the kinetochore and that this is required to maintain the SAC signal.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Regulação Fúngica da Expressão Gênica , Fosforilação/fisiologia , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
The spindle checkpoint is the prime cell-cycle control mechanism that ensures sister chromatids are bioriented before anaphase takes place. Aurora B kinase, the catalytic subunit of the chromosome passenger complex, both destabilizes kinetochore attachments that do not generate tension and simultaneously maintains the spindle checkpoint signal. However, it is unclear how the checkpoint is silenced following chromosome biorientation. We demonstrate that association of type 1 phosphatase (PP1(Dis2)) with both the N terminus of Spc7 and the nonmotor domains of the Klp5-Klp6 (kinesin-8) complex is necessary to counteract Aurora B kinase to efficiently silence the spindle checkpoint. The role of Klp5 and Klp6 in checkpoint silencing is specific to this class of kinesin and independent of their motor activities. These data demonstrate that at least two distinct pools of PP1, one kinetochore associated and the other motor associated, are needed to silence the spindle checkpoint.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Fuso Acromático/fisiologia , Sequência de Aminoácidos , Cromátides , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , Cinesinas/genética , Cinetocoros/fisiologia , Proteínas Associadas aos Microtúbulos/genética , Mitose , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/genética , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genética , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-HíbridoRESUMO
Type 1 phosphatase (PP1) antagonizes Aurora B kinase to stabilize kinetochore-microtubule attachments and to silence the spindle checkpoint. We screened for factors that exacerbate the growth defect of Δdis2 cells, which lack one of two catalytic subunits of PP1 in fission yeast, and identified Nsk1, a novel protein required for accurate chromosome segregation. During interphase, Nsk1 resides in the nucleolus but spreads throughout the nucleoplasm as cells enter mitosis. Following dephosphorylation by Clp1 (Cdc14-like) phosphatase and at least one other phosphatase, Nsk1 localizes to the interface between kinetochores and the inner face of the spindle pole body during anaphase. In the absence of Nsk1, some kinetochores become detached from spindle poles during anaphase B. If this occurs late in anaphase B, then the sister chromatids of unclustered kinetochores segregate to the correct daughter cell. These unclustered kinetochores are efficiently captured, retrieved, bioriented, and segregated during the following mitosis, as long as Dis2 is present. However, if kinetochores are detached from a spindle pole early in anaphase B, then these sister chromatids become missegregated. These data suggest Nsk1 ensures accurate chromosome segregation by promoting the tethering of kinetochores to spindle poles during anaphase B.
Assuntos
Anáfase , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Cinetocoros/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteínas de Ciclo Celular/genética , Dineínas/metabolismo , Microtúbulos/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Estabilidade Proteica , Transporte Proteico , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Fuso Acromático/genética , Fuso Acromático/metabolismoRESUMO
Phosphoinositide-3-Kinase (PI3-K) and the downstream kinases Akt and Glycogen Synthase Kinase-3 (GSK-3) have recently been implicated in regulating both microtubule (MT) dynamics and organization. Here we review the role of this signalling pathway in controlling MTs, and explore ways in which the kinases and their substrates may co-operate to spatially regulate MTs in different contexts.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Microtúbulos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Polaridade Celular , Humanos , Microtúbulos/enzimologia , Transdução de SinaisRESUMO
The chromosomal passenger complex (CPC), which is composed of conserved proteins aurora B, inner centromere protein (INCENP), survivin, and Borealin/DASRA, localizes to chromatin, kinetochores, microtubules, and the cell cortex in a cell cycle-dependent manner. The CPC is required for multiple aspects of cell division. Here we find that Drosophila melanogaster encodes two Borealin paralogues, Borealin-related (Borr) and Australin (Aust). Although Borr is a passenger in all mitotic tissues studied, it is specifically replaced by Aust for the two male meiotic divisions. We analyzed aust mutant spermatocytes to assess the effects of fully inactivating the Aust-dependent functions of the CPC. Our results indicate that Aust is required for sister chromatid cohesion, recruitment of the CPC to kinetochores, and chromosome alignment and segregation but not for meiotic histone phosphorylation or spindle formation. Furthermore, we show that the CPC is required earlier in cytokinesis than previously thought; cells lacking Aust do not initiate central spindle formation, accumulate anillin or actin at the cell equator, or undergo equatorial constriction.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Meiose/genética , Espermatócitos/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/isolamento & purificação , Segregação de Cromossomos/genética , Proteínas Contráteis/genética , Proteínas Contráteis/metabolismo , Citocinese/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/isolamento & purificação , Drosophila melanogaster/metabolismo , Drosophila melanogaster/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento/genética , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Masculino , Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação/genética , Prometáfase/genética , Transporte Proteico/genética , Espermatócitos/ultraestrutura , Fuso Acromático/genética , Fuso Acromático/metabolismo , Fuso Acromático/ultraestruturaRESUMO
Correct positioning and morphology of the mitotic spindle is achieved through regulating the interaction between microtubules (MTs) and cortical actin. Here we find that, in the Drosophila melanogaster early embryo, reduced levels of the protein kinase Akt result in incomplete centrosome migration around cortical nuclei, bent mitotic spindles, and loss of nuclei into the interior of the embryo. We show that Akt is enriched at the embryonic cortex and is required for phosphorylation of the glycogen synthase kinase-3beta homologue Zeste-white 3 kinase (Zw3) and for the cortical localizations of the adenomatosis polyposis coli (APC)-related protein APC2/E-APC and the MT + Tip protein EB1. We also show that reduced levels of Akt result in mislocalization of APC2 in postcellularized embryonic mitoses and misorientation of epithelial mitotic spindles. Together, our results suggest that Akt regulates a complex containing Zw3, Armadillo, APC2, and EB1 and that this complex has a role in stabilizing MT-cortex interactions, facilitating both centrosome separation and mitotic spindle orientation.