Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.229
Filtrar
1.
Nat Commun ; 12(1): 5931, 2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34635673

RESUMO

The chromatin remodeler RSF1 enriched at mitotic centromeres is essential for proper chromosome alignment and segregation and underlying mechanisms remain to be disclosed. We here show that PLK1 recruitment by RSF1 at centromeres creates an activating phosphorylation on Thr236 in the activation loop of Aurora B and this is indispensable for the Aurora B activation. In structural modeling the phosphorylated Thr236 enhances the base catalysis by Asp200 nearby, facilitating the Thr232 autophosphorylation. Accordingly, RSF1-PLK1 is central for Aurora B-mediated microtubule destabilization in error correction. However, under full microtubule-kinetochore attachment RSF1-PLK1 positions at kinetochores, halts activating Aurora B and phosphorylates BubR1, regardless of tension. Spatial movement of RSF1-PLK1 to kinetochores is triggered by Aurora B-mediated phosphorylation of centromeric histone H3 on Ser28. We propose a regulatory RSF1-PLK1 axis that spatiotemporally controls on/off switch on Aurora B. This feedback circuit among RSF1-PLK1-Aurora B may coordinate dynamic microtubule-kinetochore attachment in early mitosis when full tension yet to be generated.


Assuntos
Aurora Quinase B/genética , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos , Mitose , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/genética , Transativadores/genética , Ácido Aspártico/metabolismo , Aurora Quinase B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Proteínas Nucleares/deficiência , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Serina/metabolismo , Transativadores/deficiência
2.
J Cell Sci ; 134(15)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34342353

RESUMO

Centromere structure and function are defined by the epigenetic modification of histones at centromeric and pericentromeric chromatin. The constitutive heterochromatin found at pericentromeric regions is highly enriched for H3K9me3 and H4K20me3. Although mis-expression of the methyltransferase enzymes that regulate these marks, Suv39 and Suv420, is common in disease, the consequences of such changes are not well understood. Our data show that increased centromere localization of Suv39 and Suv420 suppresses centromere transcription and compromises localization of the mitotic kinase Aurora B, decreasing microtubule dynamics and compromising chromosome alignment and segregation. We find that inhibition of Suv420 methyltransferase activity partially restores Aurora B localization to centromeres and that restoration of the Aurora B-containing chromosomal passenger complex to the centromere is sufficient to suppress mitotic errors that result when Suv420 and H4K20me3 is enriched at centromeres. Consistent with a role for Suv39 and Suv420 in negatively regulating Aurora B, high expression of these enzymes corresponds with increased sensitivity to Aurora kinase inhibition in human cancer cells, suggesting that increased H3K9 and H4K20 methylation may be an underappreciated source of chromosome mis-segregation in cancer. This article has an associated First Person interview with the first author of the paper.


Assuntos
Centrômero , Cinetocoros , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Centrômero/metabolismo , Segregação de Cromossomos , Humanos , Cinetocoros/metabolismo , Mitose , Fosforilação , Transcrição Genética
3.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445523

RESUMO

The accurate segregation of chromosomes is essential for the survival of organisms and cells. Mistakes can lead to aneuploidy, tumorigenesis and congenital birth defects. The spindle assembly checkpoint ensures that chromosomes properly align on the spindle, with sister chromatids attached to microtubules from opposite poles. Here, we review how tension is used to identify and selectively destabilize incorrect attachments, and thus serves as a trigger of the spindle assembly checkpoint to ensure fidelity in chromosome segregation. Tension is generated on properly attached chromosomes as sister chromatids are pulled in opposing directions but resisted by centromeric cohesin. We discuss the role of the Aurora B kinase in tension-sensing and explore the current models for translating mechanical force into Aurora B-mediated biochemical signals that regulate correction of chromosome attachments to the spindle.


Assuntos
Aurora Quinase B/metabolismo , Cromátides/metabolismo , Cinetocoros/metabolismo , Animais , Segregação de Cromossomos , Humanos , Pontos de Checagem da Fase M do Ciclo Celular
4.
Elife ; 102021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34308839

RESUMO

Kinetochores are multi-subunit protein assemblies that link chromosomes to microtubules of the mitotic and meiotic spindle. It is still poorly understood how efficient, centromere-dependent kinetochore assembly is accomplished from hundreds of individual protein building blocks in a cell cycle-dependent manner. Here, by combining comprehensive phosphorylation analysis of native Ctf19CCAN subunits with biochemical and functional assays in the model system budding yeast, we demonstrate that Cdk1 phosphorylation activates phospho-degrons on the essential subunit Ame1CENP-U, which are recognized by the E3 ubiquitin ligase complex SCF-Cdc4. Gradual phosphorylation of degron motifs culminates in M-phase and targets the protein for degradation. Binding of the Mtw1Mis12 complex shields the proximal phospho-degron, protecting kinetochore-bound Ame1 from the degradation machinery. Artificially increasing degron strength partially suppresses the temperature sensitivity of a cdc4 mutant, while overexpression of Ame1-Okp1 is toxic in SCF mutants, demonstrating the physiological importance of this mechanism. We propose that phospho-regulated clearance of excess CCAN subunits facilitates efficient centromere-dependent kinetochore assembly. Our results suggest a novel strategy for how phospho-degrons can be used to regulate the assembly of multi-subunit complexes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas F-Box/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Divisão Celular , Centrômero/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/química , Proteínas F-Box/genética , Proteínas Associadas aos Microtúbulos/genética , Mutação de Sentido Incorreto , Organismos Geneticamente Modificados , Fosforilação , Estabilidade Proteica , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Fuso Acromático/metabolismo , Ubiquitina-Proteína Ligases/genética
5.
Dev Cell ; 56(15): 2192-2206.e8, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34331869

RESUMO

To generate haploid gametes, germ cells undergo two consecutive meiotic divisions requiring key changes to the cell division machinery. Here, we demonstrate that the protease separase rewires key cell division processes at the meiosis I/II transition by cleaving the meiosis-specific protein Meikin. Separase proteolysis does not inactivate Meikin but instead alters its function to create a distinct activity state. Full-length Meikin and the C-terminal Meikin separase cleavage product both localize to kinetochores, bind to Plk1 kinase, and promote Rec8 cleavage, but our results reveal distinct roles for these proteins in controlling meiosis. Mutations that prevent Meikin cleavage or that conditionally inactivate Meikin at anaphase I result in defective meiosis II chromosome alignment in mouse oocytes. Finally, as oocytes exit meiosis, C-Meikin is eliminated by APC/C-mediated degradation prior to the first mitotic division. Thus, multiple regulatory events irreversibly modulate Meikin activity during successive meiotic divisions to rewire the cell division machinery at two distinct transitions.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Meiose/fisiologia , Separase/metabolismo , Animais , Animais não Endogâmicos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Divisão do Núcleo Celular , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/fisiologia , Segregação de Cromossomos , Feminino , Células HeLa , Humanos , Cinetocoros/metabolismo , Camundongos , Oócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Separase/fisiologia
6.
J Cell Biol ; 220(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34081090

RESUMO

The kinetochore is the macromolecular protein complex that assembles onto centromeric DNA and binds spindle microtubules. Evolutionarily divergent kinetoplastids have an unconventional set of kinetochore proteins. It remains unknown how kinetochores assemble at centromeres in these organisms. Here, we characterize KKT2 and KKT3 in the kinetoplastid parasite Trypanosoma brucei. In addition to the N-terminal kinase domain and C-terminal divergent polo boxes, these proteins have a central domain of unknown function. We show that KKT2 and KKT3 are important for the localization of several kinetochore proteins and that their central domains are sufficient for centromere localization. Crystal structures of the KKT2 central domain from two divergent kinetoplastids reveal a unique zinc-binding domain (termed the CL domain for centromere localization), which promotes its kinetochore localization in T. brucei. Mutations in the equivalent domain in KKT3 abolish its kinetochore localization and function. Our work shows that the unique central domains play a critical role in mediating the centromere localization of KKT2 and KKT3.


Assuntos
Cinetocoros/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/genética , Relação Estrutura-Atividade , Trypanosoma brucei brucei/genética , Zinco/metabolismo
7.
J Cell Biol ; 220(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34081091

RESUMO

The step-by-step process of chromosome segregation defines the stages of the cell cycle. In eukaryotes, signals controlling these steps converge upon the kinetochore, a multiprotein assembly that connects spindle microtubules to chromosomal centromeres. Kinetochores control and adapt to major chromosomal transactions, including replication of centromeric DNA, biorientation of sister centromeres on the metaphase spindle, and transit of sister chromatids into daughter cells during anaphase. Although the mechanisms that ensure tight microtubule coupling at anaphase are at least partly understood, kinetochore adaptations that support other cell cycle transitions are not. We report here a mechanism that enables regulated control of kinetochore sumoylation. A conserved surface of the Ctf3/CENP-I kinetochore protein provides a binding site for Ulp2, the nuclear enzyme that removes SUMO chains from modified substrates. Ctf3 mutations that disable Ulp2 recruitment cause elevated inner kinetochore sumoylation and defective chromosome segregation. The location of the site within the assembled kinetochore suggests coordination between sumoylation and other cell cycle-regulated processes.


Assuntos
Segregação de Cromossomos , Cromossomos Fúngicos , Endopeptidases/metabolismo , Cinetocoros/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Microscopia Crioeletrônica , Endopeptidases/química , Endopeptidases/genética , Microscopia de Fluorescência , Modelos Moleculares , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação
8.
mBio ; 12(3): e0068721, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34128702

RESUMO

During mitosis, eukaryotic cells must duplicate and separate their chromosomes in a precise and timely manner. The apparatus responsible for this is the kinetochore, which is a large protein structure that links chromosomal DNA and spindle microtubules to facilitate chromosome alignment and segregation. The proteins that comprise the kinetochore in the protozoan parasite Trypanosoma brucei are divergent from yeast and mammals and comprise an inner kinetochore complex composed of 24 distinct proteins (KKT1 to KKT23, KKT25) that include four protein kinases, CLK1 (KKT10), CLK2 (KKT19), KKT2, and KKT3. We recently reported the identification of a specific trypanocidal inhibitor of T. brucei CLK1, an amidobenzimidazole, AB1. We now show that chemical inhibition of CLK1 with AB1 impairs inner kinetochore recruitment and compromises cell cycle progression, leading to cell death. Here, we show that KKT2 is a substrate for CLK1 and identify phosphorylation of S508 by CLK1 to be essential for KKT2 function and for kinetochore assembly. Additionally, KKT2 protein kinase activity is required for parasite proliferation but not for assembly of the inner kinetochore complex. We also show that chemical inhibition of the aurora kinase AUK1 does not affect CLK1 phosphorylation of KKT2, indicating that AUK1 and CLK1 are in separate regulatory pathways. We propose that CLK1 is part of a divergent signaling cascade that controls kinetochore function via phosphorylation of the inner kinetochore protein kinase KKT2. IMPORTANCE In eukaryotic cells, kinetochores are large protein complexes that link chromosomes to dynamic microtubule tips, ensuring proper segregation and genomic stability during cell division. Several proteins tightly coordinate kinetochore functions, including the protein kinase aurora kinase B. The kinetochore has diverse evolutionary roots. For example, trypanosomatids, single-cell parasitic protozoa that cause several neglected tropical diseases, possess a unique repertoire of kinetochore components whose regulation during the cell cycle remains unclear. Here, we shed light on trypanosomatid kinetochore biology by showing that the protein kinase CLK1 coordinates the assembly of the inner kinetochore by phosphorylating one of its components, KKT2, allowing the timely spatial recruitment of the rest of the kinetochore proteins and posterior attachment to microtubules in a process that is aurora kinase B independent.


Assuntos
Regulação da Expressão Gênica , Cinetocoros/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Mitose , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteínas de Protozoários/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
9.
Int J Mol Sci ; 22(9)2021 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-34063622

RESUMO

Ribonucleic acid export 1 (Rae1) is an important nucleoporin that participates in mRNA export during the interphase of higher eukaryotes and regulates the mitotic cell cycle. In this study, small RNA interference technology was used to knockdown Rae1, and immunofluorescence, immunoblotting, and chromosome spreading were used to study the role of Rae1 in mouse oocyte meiotic maturation. We found that Rae1 is a crucial regulator of meiotic maturation of mouse oocytes. After the resumption of meiosis (GVBD), Rae1 was concentrated on the kinetochore structure. The knockdown of Rae1 by a specific siRNA inhibited GVBD progression at 2 h, finally leading to a decreased 14 h polar body extrusion (PBE) rate. However, a comparable 14 h PBE rate was found in the control, and the Rae1 knockdown groups that had already undergone GVBD. Furthermore, we found elevated PBE after 9.5 h in the Rae1 knockdown oocytes. Further analysis revealed that Rae1 depletion significantly decreased the protein level of securin. In addition, we detected weakened kinetochore-microtubule (K-MT) attachments, misaligned chromosomes, and an increased incidence of aneuploidy in the Rae1 knockdown oocytes. Collectively, we propose that Rae1 modulates securin protein levels, which contribute to chromosome alignment, K-MT attachments, and aneuploidy in meiosis.


Assuntos
Meiose/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas à Matriz Nuclear/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Oócitos/metabolismo , Animais , Técnicas de Silenciamento de Genes , Técnicas de Maturação in Vitro de Oócitos , Cinetocoros/metabolismo , Camundongos , Oócitos/crescimento & desenvolvimento , Corpos Polares/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética
10.
Sci Rep ; 11(1): 11165, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34045512

RESUMO

The spindle and kinetochore-associated protein complex (Ska) is an essential component in chromosome segregation. It comprises three proteins (Ska1, Ska2, and Ska3) with theorized roles in chromosomal instability and tumor development, and its overexpression has been widely reported in a variety of tumors. However, the prognostic significance and immune infiltration of Ska proteins in hepatocellular carcinoma (HCC) are not completely understood. The bioinformatics tools Oncomine, UALCAN, gene expression profiling interactive analysis 2 (GEPIA2), cBioPortal, GeneMANIA, Metascape, and TIMER were used to analyze differential expression, prognostic value, genetic alteration, and immune cell infiltration of the Ska protein complex in HCC patients. We found that the mRNA expression of the Ska complex was markedly upregulated in HCC. High expression of the Ska complex is closely correlated with tumor stage, patient race, tumor grade, and TP53 mutation status. In addition, high expression of the Ska complex was significantly correlated with poor disease-free survival, while the high expression levels of Ska1 and Ska3 were associated with shorter overall survival. The biological functions of the Ska complex in HCC primarily involve the amplification of signals from kinetochores, the mitotic spindle, and (via a MAD2 invasive signal) unattached kinetochores. Furthermore, the expression of the complex was positively correlated with tumor-infiltrating cells. These results may provide new insights into the development of immunotherapeutic targets and prognostic biomarkers for HCC.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Cinetocoros/metabolismo , Neoplasias Hepáticas/genética , Proteínas Associadas aos Microtúbulos/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Prognóstico , Fuso Acromático , Taxa de Sobrevida
11.
PLoS Genet ; 17(5): e1009592, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34033659

RESUMO

The spindle assembly checkpoint (SAC) prevents anaphase onset in response to chromosome attachment defects, and SAC silencing is essential for anaphase onset. Following anaphase onset, activated Cdc14 phosphatase dephosphorylates the substrates of cyclin-dependent kinase to facilitate anaphase progression and mitotic exit. In budding yeast, Cdc14 dephosphorylates Fin1, a regulatory subunit of protein phosphatase 1 (PP1), to enable kinetochore localization of Fin1-PP1. We previously showed that kinetochore-localized Fin1-PP1 promotes the removal of the SAC protein Bub1 from the kinetochore during anaphase. We report here that Fin1-PP1 also promotes kinetochore removal of Bub3, the Bub1 partner, but has no effect on another SAC protein Mad1. Moreover, the kinetochore localization of Bub1-Bub3 during anaphase requires Aurora B/Ipl1 kinase activity. We further showed that Fin1-PP1 facilitates the dephosphorylation of kinetochore protein Ndc80, a known Ipl1 substrate. This dephosphorylation reduces kinetochore association of Bub1-Bub3 during anaphase. In addition, we found that untimely Ndc80 dephosphorylation causes viability loss in response to tensionless chromosome attachments. These results suggest that timely localization of Fin1-PP1 to the kinetochore controls the functional window of SAC and is therefore critical for faithful chromosome segregation.


Assuntos
Anáfase , Aurora Quinases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Segregação de Cromossomos , Cinetocoros/química , Cinetocoros/efeitos dos fármacos , Viabilidade Microbiana/genética , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fosforilação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fuso Acromático/efeitos dos fármacos , Fatores de Tempo
12.
EMBO Rep ; 22(7): e52242, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34013668

RESUMO

During metaphase, in response to improper kinetochore-microtubule attachments, the spindle assembly checkpoint (SAC) activates the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex/cyclosome (APC/C). This process is orchestrated by the kinase Mps1, which initiates the assembly of the MCC onto kinetochores through a sequential phosphorylation-dependent signalling cascade. The Mad1-Mad2 complex, which is required to catalyse MCC formation, is targeted to kinetochores through a direct interaction with the phosphorylated conserved domain 1 (CD1) of Bub1. Here, we present the crystal structure of the C-terminal domain of Mad1 (Mad1CTD ) bound to two phosphorylated Bub1CD1 peptides at 1.75 Å resolution. This interaction is mediated by phosphorylated Bub1 Thr461, which not only directly interacts with Arg617 of the Mad1 RLK (Arg-Leu-Lys) motif, but also directly acts as an N-terminal cap to the CD1 α-helix dipole. Surprisingly, only one Bub1CD1 peptide binds to the Mad1 homodimer in solution. We suggest that this stoichiometry is due to inherent asymmetry in the coiled-coil of Mad1CTD and has implications for how the Mad1-Bub1 complex at kinetochores promotes efficient MCC assembly.


Assuntos
Proteínas de Ciclo Celular , Cinetocoros , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Cinetocoros/metabolismo , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Fosforilação , Transdução de Sinais , Fuso Acromático/metabolismo
13.
J Cell Biol ; 220(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33851957

RESUMO

To establish chromosome biorientation, aberrant kinetochore-microtubule interaction must be resolved (error correction) by Aurora B kinase. Aurora B differentially regulates kinetochore attachment to the microtubule plus end and its lateral side (end-on and lateral attachment, respectively). However, it is still unclear how kinetochore-microtubule interactions are exchanged during error correction. Here, we reconstituted the budding yeast kinetochore-microtubule interface in vitro by attaching the Ndc80 complexes to nanobeads. These Ndc80C nanobeads recapitulated in vitro the lateral and end-on attachments of authentic kinetochores on dynamic microtubules loaded with the Dam1 complex. This in vitro assay enabled the direct comparison of lateral and end-on attachment strength and showed that Dam1 phosphorylation by Aurora B makes the end-on attachment weaker than the lateral attachment. Similar reconstitutions with purified kinetochore particles were used for comparison. We suggest the Dam1 phosphorylation weakens interaction with the Ndc80 complex, disrupts the end-on attachment, and promotes the exchange to a new lateral attachment, leading to error correction.


Assuntos
Aurora Quinase B/metabolismo , Cinetocoros/fisiologia , Microtúbulos/fisiologia , Mitose , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Aurora Quinase B/genética , Cinetocoros/metabolismo , Mutação , Proteínas Nucleares/genética , Fosforilação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
14.
Nucleic Acids Res ; 49(16): 9154-9173, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-33872374

RESUMO

Foreign DNA microinjected into the Caenorhabditis elegans syncytial gonad forms episomal extra-chromosomal arrays, or artificial chromosomes (ACs), in embryos. Short, linear DNA fragments injected concatemerize into high molecular weight (HMW) DNA arrays that are visible as punctate DAPI-stained foci in oocytes, and they undergo chromatinization and centromerization in embryos. The inner centromere, inner kinetochore and spindle checkpoint components, including AIR-2, CENP-AHCP-3, Mis18BP1KNL-2 and BUB-1, respectively, assemble onto the nascent ACs during the first mitosis. The DNA replication efficiency of ACs improves over several cell cycles, which correlates with the improvement of kinetochore bi-orientation and proper segregation of ACs. Depletion of condensin II subunits, like CAPG-2 and SMC-4, but not the replicative helicase component, MCM-2, reduces de novo CENP-AHCP-3 level on nascent ACs. Furthermore, H3K9ac, H4K5ac and H4K12ac are highly enriched on newly chromatinized ACs. RbAp46/48LIN-53 and HAT-1, which affect the acetylation of histone H3 and H4, are essential for chromatinization, de novo centromere formation and segregation competency of nascent ACs. RbAp46/48LIN-53 or HAT-1 depletion causes the loss of both CENP-AHCP-3 and Mis18BP1KNL-2 initial deposition at de novo centromeres on ACs. This phenomenon is different from centromere maintenance on endogenous chromosomes, where Mis18BP1KNL-2 functions upstream of RbAp46/48LIN-53.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Cromossomos Artificiais/genética , Histona Acetiltransferases/metabolismo , Cinetocoros/metabolismo , Proteínas Repressoras/metabolismo , Acetilação , Animais , Caenorhabditis elegans , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA , Código das Histonas , Histonas/metabolismo , Componente 2 do Complexo de Manutenção de Minicromossomo/metabolismo , Mitose
15.
Int J Mol Sci ; 22(6)2021 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-33804687

RESUMO

Cell proliferation and sexual reproduction require the faithful segregation of chromosomes. Chromosome segregation is driven by the interaction of chromosomes with the spindle, and the attachment of chromosomes to the proper spindle poles is essential. Initial attachments are frequently erroneous due to the random nature of the attachment process; however, erroneous attachments are selectively eliminated. Proper attachment generates greater tension at the kinetochore than erroneous attachments, and it is thought that attachment selection is dependent on this tension. However, studies of meiotic chromosome segregation suggest that attachment elimination cannot be solely attributed to tension, and the precise mechanism of selective elimination of erroneous attachments remains unclear. During attachment elimination, chromosomes oscillate between the spindle poles. A recent study on meiotic chromosome segregation in fission yeast has suggested that attachment elimination is coupled to chromosome oscillation. In this review, the possible contribution of chromosome oscillation in the elimination of erroneous attachment is discussed in light of the recent finding.


Assuntos
Segregação de Cromossomos , Cinetocoros/metabolismo , Meiose , Microtúbulos/metabolismo , Mitose , Animais , Centrômero/genética , Centrômero/metabolismo , Humanos , Modelos Biológicos , Ligação Proteica , Fuso Acromático
16.
EMBO J ; 40(7): e106797, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33644892

RESUMO

Partitioning of the genome in meiosis occurs through two highly specialized cell divisions, named meiosis I and meiosis II. Step-wise cohesin removal is required for chromosome segregation in meiosis I, and sister chromatid segregation in meiosis II. In meiosis I, mono-oriented sister kinetochores appear as fused together when examined by high-resolution confocal microscopy, whereas they are clearly separated in meiosis II, when attachments are bipolar. It has been proposed that bipolar tension applied by the spindle is responsible for the physical separation of sister kinetochores, removal of cohesin protection, and chromatid separation in meiosis II. We show here that this is not the case, and initial separation of sister kinetochores occurs already in anaphase I independently of bipolar spindle forces applied on sister kinetochores, in mouse oocytes. This kinetochore individualization depends on separase cleavage activity. Crucially, without kinetochore individualization in meiosis I, bivalents when present in meiosis II oocytes separate into chromosomes and not sister chromatids. This shows that whether centromeric cohesin is removed or not is determined by the kinetochore structure prior to meiosis II.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Meiose , Animais , Células Cultivadas , Cromátides/genética , Cromátides/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/metabolismo
17.
EMBO J ; 40(7): e106812, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33644894

RESUMO

Genome haploidization involves sequential loss of cohesin from chromosome arms and centromeres during two meiotic divisions. At centromeres, cohesin's Rec8 subunit is protected from separase cleavage at meiosis I and then deprotected to allow its cleavage at meiosis II. Protection of centromeric cohesin by shugoshin-PP2A seems evolutionarily conserved. However, deprotection has been proposed to rely on spindle forces separating the Rec8 protector from cohesin at metaphase II in mammalian oocytes and on APC/C-dependent destruction of the protector at anaphase II in yeast. Here, we have activated APC/C in the absence of sister kinetochore biorientation at meiosis II in yeast and mouse oocytes, and find that bipolar spindle forces are dispensable for sister centromere separation in both systems. Furthermore, we show that at least in yeast, protection of Rec8 by shugoshin and inhibition of separase by securin are both required for the stability of centromeric cohesin at metaphase II. Our data imply that related mechanisms preserve the integrity of dyad chromosomes during the short metaphase II of yeast and the prolonged metaphase II arrest of mammalian oocytes.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Meiose , Animais , Células Cultivadas , Feminino , Camundongos , Oócitos/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
Plant Cell ; 33(1): 27-43, 2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33751090

RESUMO

The bipolar mitotic spindle is a highly conserved structure among eukaryotes that mediates chromosome alignment and segregation. Spindle assembly and size control are facilitated by force-generating microtubule-dependent motor proteins known as kinesins. In animals, kinesin-12 cooperates with kinesin-5 to produce outward-directed forces necessary for spindle assembly. In plants, the relevant molecular mechanisms for spindle formation are poorly defined. While an Arabidopsis thaliana kinesin-5 ortholog has been identified, the kinesin-12 ortholog in plants remains elusive. In this study, we provide experimental evidence for the function of Arabidopsis KINESIN-12E in spindle assembly. In kinesin-12e mutants, a delay in spindle assembly is accompanied by the reduction of spindle size, demonstrating that KINESIN-12E contributes to mitotic spindle architecture. Kinesin-12E localization is mitosis-stage specific, beginning with its perinuclear accumulation during prophase. Upon nuclear envelope breakdown, KINESIN-12E decorates subpopulations of microtubules in the spindle and becomes progressively enriched in the spindle midzone. Furthermore, during cytokinesis, KINESIN-12E shares its localization at the phragmoplast midzone with several functionally diversified Arabidopsis KINESIN-12 members. Changes in the kinetochore and in prophase and metaphase spindle dynamics occur in the absence of KINESIN-12E, suggest it might play an evolutionarily conserved role during spindle formation similar to its spindle-localized animal kinesin-12 orthologs.


Assuntos
Arabidopsis/metabolismo , Microtúbulos/metabolismo , Cinesina/metabolismo , Cinetocoros/metabolismo , Metáfase/fisiologia , Prófase/fisiologia
19.
Nat Commun ; 12(1): 1763, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741944

RESUMO

Accurate chromosome segregation relies on the specific centromeric nucleosome-kinetochore interface. In budding yeast, the centromere CBF3 complex guides the deposition of CENP-A, an H3 variant, to form the centromeric nucleosome in a DNA sequence-dependent manner. Here, we determine the structures of the centromeric nucleosome containing the native CEN3 DNA and the CBF3core bound to the canonical nucleosome containing an engineered CEN3 DNA. The centromeric nucleosome core structure contains 115 base pair DNA including a CCG motif. The CBF3core specifically recognizes the nucleosomal CCG motif through the Gal4 domain while allosterically altering the DNA conformation. Cryo-EM, modeling, and mutational studies reveal that the CBF3core forms dynamic interactions with core histones H2B and CENP-A in the CEN3 nucleosome. Our results provide insights into the structure of the budding yeast centromeric nucleosome and the mechanism of its assembly, which have implications for analogous processes of human centromeric nucleosome formation.


Assuntos
Centrômero/metabolismo , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Cinetocoros/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Centrômero/genética , Centrômero/ultraestrutura , Proteína Centromérica A/química , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Microscopia Crioeletrônica , DNA Fúngico/química , DNA Fúngico/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Cinetocoros/química , Conformação de Ácido Nucleico , Nucleossomos/genética , Nucleossomos/ultraestrutura , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos
20.
J Cell Biol ; 220(4)2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33620383

RESUMO

Chromosomal instability (CIN) is a hallmark of many cancers. Restricting the localization of centromeric histone H3 variant CENP-A to centromeres prevents CIN. CENP-A overexpression (OE) and mislocalization have been observed in cancers and correlate with poor prognosis; however, the molecular consequences of CENP-A OE on CIN and aneuploidy have not been defined. Here, we show that CENP-A OE leads to its mislocalization and CIN with lagging chromosomes and micronuclei in pseudodiploid DLD1 cells and xenograft mouse model. CIN is due to reduced localization of proteins to the kinetochore, resulting in defects in kinetochore integrity and unstable kinetochore-microtubule attachments. CENP-A OE contributes to reduced expression of cell adhesion genes and higher invasion of DLD1 cells. We show that CENP-A OE contributes to aneuploidy with karyotypic heterogeneity in human cells and xenograft mouse model. In summary, our results provide a molecular link between CENP-A OE and aneuploidy, and suggest that karyotypic heterogeneity may contribute to the aggressive phenotype of CENP-A-overexpressing cancers.


Assuntos
Aneuploidia , Proteína Centromérica A/biossíntese , Instabilidade Cromossômica , Cinetocoros/metabolismo , Micronúcleos com Defeito Cromossômico , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Animais , Linhagem Celular Tumoral , Proteína Centromérica A/genética , Xenoenxertos , Humanos , Cinetocoros/patologia , Camundongos , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Neoplasias/genética , Neoplasias/patologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...