RESUMO
The composition and structure of centromeric nucleosomes, which contain the histone H3 variant CENP-A, is intensely debated. Two independent studies in this issue, in yeast and human cells, now suggest that CENP-A nucleosomes adopt different structures depending on the stage of the cell cycle.
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Accurate chromosome segregation requires the spindle assembly checkpoint to be active at the onset of mitosis, before being silenced following chromosome alignment. p31(comet) is a checkpoint antagonist in that its inhibition delays mitotic exit, whereas its overexpression overrides the checkpoint. How exactly p31(comet) antagonises the checkpoint is unclear. A prevalent model is that p31(comet) acts as a 'cap' by inhibiting recruitment of the open conformation form of Mad2 (O-Mad2) to the kinetochore-bound complex of Mad1-C-Mad2 (closed conformation Mad2), an essential step that is required for checkpoint activation. Here, we show that although p31(comet) localises to kinetochores in mitosis, modulation of its activity has no effect on recruitment of O-Mad2 to kinetochores. Rather, our observations support a checkpoint-silencing role for p31(comet) downstream of kinetochores. We show that p31(comet) binds Mad2 when it is bound to the mitotic checkpoint complex (MCC) components BubR1 and Cdc20. Furthermore, RNAi-mediated inhibition of p31(comet) results in more Mad2 bound to BubR1-Cdc20, and conversely, overexpression of p31(comet) results in less Mad2 bound to BubR1-Cdc20. Addition of recombinant p31(comet) to checkpoint-arrested extracts removes Mad2 from the MCC, whereas a p31(comet) mutant that cannot bind Mad2 has no effect. Significantly, expression of a Mad2 mutant that cannot bind p31(comet) prolongs the metaphase to anaphase transition. Taken together, our data support the notion that p31(comet) negatively regulates the spindle assembly checkpoint by extracting Mad2 from the MCC.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células/citologia , Mitose , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ligação ao Cálcio/genética , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/genética , Linhagem Celular , Células/metabolismo , Humanos , Cinetocoros/metabolismo , Proteínas Mad2 , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Repressoras/genética , Fuso Acromático/genética , Fuso Acromático/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Noradrenergic projections from the brainstem locus coeruleus drive arousal, attentiveness, mood, and memory, but specific adrenoceptor (AR) function across the varied brain cell types has not been extensively characterized, especially with agonists. This study reports a pharmacological analysis of brain AR function, offering insights for innovative therapeutic interventions that might serve to compensate for locus coeruleus decline, known to develop in the earliest phases of neurodegenerative diseases. First, ß-AR agonist activities were measured in recombinant cell systems and compared with those of isoprenaline to generate Δlog(Emax/EC50) values, system-independent metrics of agonist activity, that, in turn, provide receptor subtype fingerprints. These fingerprints were then used to assess receptor subtype expression across human brain cell systems and compared with Δlog(Emax/EC50) values arising from ß-arrestin activation or measurements of cAMP response desensitization to assess the possibility of ligand bias among ß-AR agonists. Agonist activity profiles were confirmed to be system-independent and, in particular, revealed ß2-AR functional expression across several human brain cell types. Broad ß2-AR function observed is consistent with noradrenergic tone arising from the locus coeruleus exerting heterocellular neuroexcitatory and homeostatic influence. Notably, Δlog(Emax/EC50) measurements suggest that tested ß-AR agonists do not show ligand bias as it pertains to homologous receptor desensitization in the system examined. Δlog(Emax/EC50) agonist fingerprinting is a powerful means of assessing receptor subtype expression regardless of receptor expression levels or assay readout, and the method may be applicable to future use for novel ligands and tissues expressing any receptor with available reference agonists.
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The spindle checkpoint restrains anaphase onset and mitotic exit until all chromosomes are stably attached to the mitotic spindle via their kinetochores. The Tao1 protein kinase was recently reported as a novel spindle checkpoint component. When an siRNA was used to repress Tao1, the essential spindle checkpoint component Mad2 failed to localise to kinetochores, and cells rapidly exited mitosis. Tao1 was also shown to interact with BubR1, another essential checkpoint component, and be rapidly degraded after mitosis, a feature typical of many mitotic regulators. Here, we identify four different siRNAs that repress Tao1 protein levels as efficiently as the previously reported siRNA. However, these siRNAs do not override the spindle checkpoint. We also present data indicating that Tao1 does not interact with BubR1 and that it is not rapidly degraded after mitosis. We show that the previously reported siRNA not only represses Tao1 but also dramatically reduces Mad2 protein levels. Crucially, expression of exogenous Mad2, but not Tao1, rescued the spindle checkpoint phenotype induced by this siRNA. Thus, the key functional data implicating Tao1 in the spindle checkpoint can be explained by an off-target siRNA phenomenon that results in Mad2 inhibition. Taken together, our data do not support the notion that Tao1 is a component of the spindle checkpoint.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Expressão Gênica , MAP Quinase Quinase Quinases/metabolismo , Mitose , Fuso Acromático/metabolismo , Anáfase , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Cdc20 , Proteínas de Ciclo Celular/genética , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Células HeLa , Humanos , Cinetocoros/metabolismo , Proteínas Mad2 , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Repressoras/metabolismo , Fuso Acromático/genéticaRESUMO
Vertebrate centromeres are epigenetically defined by nucleosomes containing the histone H3 variant, CENP-A. CENP-A nucleosome assembly requires the three-protein Mis18 complex (Mis18α, Mis18ß, and M18BP1) that recruits the CENP-A chaperone HJURP to centromeres, but how the Mis18 complex recognizes centromeric chromatin is unknown. Using Xenopus egg extract, we show that direct, cell-cycle-regulated binding of M18BP1 to CENP-A nucleosomes recruits the Mis18 complex to interphase centromeres to promote new CENP-A nucleosome assembly. We demonstrate that Xenopus M18BP1 binds CENP-A nucleosomes using a motif that is widely conserved except in mammals. The M18BP1 motif resembles a CENP-A nucleosome binding motif in CENP-C, and we show that CENP-C competes with M18BP1 for CENP-A nucleosome binding at centromeres. We show that both CENP-C and M18BP1 recruit HJURP to centromeres for new CENP-A assembly. This study defines cellular mechanisms for recruiting CENP-A assembly factors to existing CENP-A nucleosomes for the epigenetic inheritance of centromeres.
Assuntos
Autoantígenos/metabolismo , Proteínas de Transporte/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Nucleossomos/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Proteína Centromérica A , Complexos Multiproteicos/metabolismo , Ligação ProteicaRESUMO
Faithful chromosome segregation is accomplished by attachment of chromosomes to spindle microtubules using the kinetochore. In a major step forward in understanding the functional and structural complexity of kinetochores, a 21-subunit human centromere - kinetochore complex has been reconstituted entirely from purified components, recreating the connection between DNA and microtubule.
Assuntos
Segregação de Cromossomos/fisiologia , Cinetocoros/fisiologia , Mitose/fisiologia , Eucariotos/citologia , MicrotúbulosRESUMO
Centromeres are specified epigenetically through the deposition of the centromere-specific histone H3 variant CENP-A. However, how additional epigenetic features are involved in centromere specification is unknown. Here, we find that histone H4 Lys5 and Lys12 acetylation (H4K5ac and H4K12ac) primarily occur within the pre-nucleosomal CENP-A-H4-HJURP (CENP-A chaperone) complex, before centromere deposition. We show that H4K5ac and H4K12ac are mediated by the RbAp46/48-Hat1 complex and that RbAp48-deficient DT40 cells fail to recruit HJURP to centromeres and do not incorporate new CENP-A at centromeres. However, C-terminally-truncated HJURP, that does not bind CENP-A, does localize to centromeres in RbAp48-deficient cells. Acetylation-dead H4 mutations cause mis-localization of the CENP-A-H4 complex to non-centromeric chromatin. Crucially, CENP-A with acetylation-mimetic H4 was assembled specifically into centromeres even in RbAp48-deficient DT40 cells. We conclude that H4K5ac and H4K12ac, mediated by RbAp46/48, facilitates efficient CENP-A deposition into centromeres.
Assuntos
Proteína Centromérica A/metabolismo , Centrômero/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Nucleossomos/metabolismo , Acetilação , Animais , Linhagem Celular Tumoral , Centrômero/genética , Proteína Centromérica A/genética , Galinhas , Cromatina/metabolismo , Epigênese Genética , Histonas/genética , Humanos , Lisina/metabolismo , Chaperonas Moleculares/genética , Mutação , Nucleossomos/genética , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Proteína 7 de Ligação ao Retinoblastoma/metabolismoRESUMO
Centromeres are defined by the presence of CENP-A nucleosomes in chromatin and are essential for accurate chromosome segregation. Centromeric chromatin epigenetically seeds new CENP-A nucleosome formation, thereby maintaining functional centromeres as cells divide. The features within centromeric chromatin that direct new CENP-A assembly remain unclear. Here, we developed a cell-free CENP-A assembly system that enabled the study of chromatin-bound CENP-A and soluble CENP-A separately. We show that two distinct domains of CENP-A within existing CENP-A nucleosomes are required for new CENP-A assembly and that CENP-A nucleosomes recruit the CENP-A assembly factors CENP-C and M18BP1 independently. Furthermore, we demonstrate that the mechanism of CENP-C recruitment to centromeres is dependent on the density of underlying CENP-A nucleosomes.
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Autoantígenos/metabolismo , Centrômero/metabolismo , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Animais , Autoantígenos/química , Proteínas de Transporte/metabolismo , Divisão Celular , Sistema Livre de Células , Proteína Centromérica A , Cromatina/química , Proteínas Cromossômicas não Histona/química , Segregação de Cromossomos , Histonas/metabolismo , Humanos , Nucleossomos/genética , Estrutura Terciária de Proteína , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
A fundamental challenge for the survival of all organisms is maintaining the integrity of the genome in all cells. Cells must therefore segregate their replicated genome equally during each cell division. Eukaryotic organisms package their genome into a number of physically distinct chromosomes, which replicate during S phase and condense during prophase of mitosis to form paired sister chromatids. During mitosis, cells form a physical connection between each sister chromatid and microtubules of the mitotic spindle, which segregate one copy of each chromatid to each new daughter cell. The centromere is the DNA locus on each chromosome that creates the site of this connection. In this review, we present a brief history of centromere research and discuss our current knowledge of centromere establishment, maintenance, composition, structure, and function in mitosis.
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Autoantígenos/metabolismo , Centrômero/fisiologia , Centrômero/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/fisiologia , Epigênese Genética/fisiologia , Cinetocoros/metabolismo , Mitose/fisiologia , Proteína Centromérica A , HumanosRESUMO
Centromeres play essential roles in equal chromosome segregation by directing the assembly of the microtubule binding kinetochore and serving as the cohesion site between sister chromatids. Here, we review the significant recent progress in our understanding of centromere protein assembly and how centromere proteins form the foundation of the kinetochore.
Assuntos
Centrômero/metabolismo , Segregação de Cromossomos , Cinetocoros/metabolismo , Animais , Cromátides/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Microtúbulos/metabolismo , Ligação ProteicaRESUMO
During mitosis and meiosis, the spindle assembly checkpoint acts to maintain genome stability by delaying cell division until accurate chromosome segregation can be guaranteed. Accuracy requires that chromosomes become correctly attached to the microtubule spindle apparatus via their kinetochores. When not correctly attached to the spindle, kinetochores activate the spindle assembly checkpoint network, which in turn blocks cell cycle progression. Once all kinetochores become stably attached to the spindle, the checkpoint is inactivated, which alleviates the cell cycle block and thus allows chromosome segregation and cell division to proceed. Here we review recent progress in our understanding of how the checkpoint signal is generated, how it blocks cell cycle progression and how it is extinguished.