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1.
Cell Prolif ; 52(5): e12657, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31264311

RESUMO

OBJECTIVES: A high rate of chromosome aneuploidy is exhibited in in vitro fertilization (IVF)-derived embryos. Our previous experiments suggested that reactive oxygen species (ROS) can activate Mad2, a key protein in the spindle assembly checkpoint (SAC), and delay the first mitotic, providing time to prevent the formation of embryonic aneuploidy. We aimed to determine whether mitotic kinase Aurora B was involved in the SAC function to prevent aneuploidy in IVF-derived embryos. MATERIALS AND METHODS: We analysed aneuploidy formation and repair during embryo pre-implantation via 4',6-diamidino-2-phenylindole (DAPI) staining and karyotype analysis. We assessed Aurora B activation by immunofluorescence and investigated the effect of Aurora B inhibition on embryo injury-related variables, such as embryonic development, ROS levels, mitochondrial membrane potential and γH2AX-positive expression. RESULTS: We observed the expression and phosphorylation of Thr232 in Aurora B in oxidative stress-induced zygotes. Moreover, inhibition of Aurora B caused chromosome mis-segregation, abnormal spindle structures, abnormal chromosome number and reduced expression of Mad2 in IVF embryos. Our results suggest that Aurora B causes mitotic arrest and participates in SAC via Mad2 and H3S10P, which is required for self-correction of aneuploidies. CONCLUSIONS: We demonstrate here that oxidative stress-induced DNA damage triggers Aurora B-mediated activation of SAC, which prevents aneuploidy at the first mitotic cleavage in early mouse IVF embryos.


Assuntos
Aurora Quinase B/metabolismo , Proteínas Mad2/metabolismo , Aneuploidia , Animais , Aurora Quinase B/antagonistas & inibidores , Segregação de Cromossomos/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Feminino , Peróxido de Hidrogênio/farmacologia , Pontos de Checagem da Fase M do Ciclo Celular , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitose , Organofosfatos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Quinazolinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Zigoto/metabolismo
2.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 41(3): 419-424, 2019 Jun 30.
Artigo em Chinês | MEDLINE | ID: mdl-31282340

RESUMO

The chromosomal aneuploidy in oocytes is one of main causes of abortion and neonatal birth defects.It is mainly due to the premature separation of sister chromatid caused by the loss of Cohesin protein complex and the non-disjunction sister chromatids caused by abnormal microtubule dynamics aneuploidy.As a pathway of protein post-translational modification,SUMO modification(or SUMOylation)involves many physiological regulation processes including cell proliferation,differentiation,apoptosis,and cycle regulation.In the oocytes,SUMOylation can regulate the localization of Cohesin protein complex on the chromosome to affect the chromosomal aneuploidy in oocytes caused by premature separation of sister chromatid.On the other hand,SUMOylation can regulate the microtubule dynamics to affect the chromosomal aneuploidy in oocytes caused by non-disjunction sister chromatids.Therefore,SUMOylation plays an important role in regulating the chromosomal aneuploidy of oocytes;the exact mechanisms via which the SUMOylated substrates affect aneuploidy in oocytes remain unclear.This articles reviews the roles of SUMOylation in premature separation and non-isolated chromatid aneuploidy in oocyte from the effects of SUMOylationon Cohesin protein complex and microtubule dynamics.


Assuntos
Aneuploidia , Cromátides , Segregação de Cromossomos , Oócitos/citologia , Sumoilação , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Humanos , Microtúbulos
3.
Nat Commun ; 10(1): 2861, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253795

RESUMO

Centromeres provide a pivotal function for faithful chromosome segregation. They serve as a foundation for the assembly of the kinetochore complex and spindle connection, which is essential for chromosome biorientation. Cells lacking Polo-like kinase 1 (PLK1) activity suffer severe chromosome alignment defects, which is believed primarily due to unstable kinetochore-microtubule attachment. Here, we reveal a previously undescribed mechanism named 'centromere disintegration' that drives chromosome misalignment in PLK1-inactivated cells. We find that PLK1 inhibition does not necessarily compromise metaphase establishment, but instead its maintenance. We demonstrate that this is caused by unlawful unwinding of DNA by BLM helicase at a specific centromere domain underneath kinetochores. Under bipolar spindle pulling, the distorted centromeres are promptly decompacted into DNA threadlike molecules, leading to centromere rupture and whole-chromosome arm splitting. Consequently, chromosome alignment collapses. Our study unveils an unexpected role of PLK1 as a chromosome guardian to maintain centromere integrity for chromosome biorientation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fuso Acromático/fisiologia , Linhagem Celular , Pareamento Cromossômico/fisiologia , Humanos , Cinetocoros , Interferência de RNA , Timidina/farmacologia
4.
Nat Commun ; 10(1): 2862, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253793

RESUMO

DNA double strand breaks (DSBs) pose a high risk for genome integrity. Cells repair DSBs through homologous recombination (HR) when a sister chromatid is available. HR is upregulated by the cycling dependent kinase (CDK) despite the paradox of telophase, where CDK is high but a sister chromatid is not nearby. Here we study in the budding yeast the response to DSBs in telophase, and find they activate the DNA damage checkpoint (DDC), leading to a telophase-to-G1 delay. Outstandingly, we observe a partial reversion of sister chromatid segregation, which includes approximation of segregated material, de novo formation of anaphase bridges, and coalescence between sister loci. We finally show that DSBs promote a massive change in the dynamics of telophase microtubules (MTs), together with dephosphorylation and relocalization of kinesin-5 Cin8. We propose that chromosome segregation is not irreversible and that DSB repair using the sister chromatid is possible in telophase.


Assuntos
Cromátides/metabolismo , Segregação de Cromossomos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Fúngico/genética , Troca de Cromátide Irmã , Telófase/genética , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Recombinação Genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Biochim Biophys Acta Rev Cancer ; 1872(1): 60-65, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31152819

RESUMO

Hepatocellular carcinoma (HCC), the most common form of liver cancer, represents a health problem in hepatic viruses-eradicating era because obesity, type 2 diabetes, and nonalcoholic steatohepatitis (NASH) are considered emerging pathogenic factors. Metabolic disorders underpin mitotic errors that lead to numerical and structural chromosome aberrations in a significant proportion of cell divisions. Here, we review that genomically unstable HCCs show evidence for a paradoxically DNA damage response (DDR) which leads to ongoing chromosome segregation errors. The understanding of DDR induced by defective mitoses is crucial to our ability to develop or improve liver cancer therapeutic strategies.


Assuntos
Carcinoma Hepatocelular/genética , Genoma Humano/genética , Instabilidade Genômica/genética , Neoplasias Hepáticas/genética , Carcinoma Hepatocelular/patologia , Instabilidade Cromossômica/genética , Segregação de Cromossomos/genética , Dano ao DNA/genética , Humanos , Neoplasias Hepáticas/patologia , Mitose/genética
6.
Mol Cell ; 74(5): 866-876, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31173722

RESUMO

The replisome quickly and accurately copies billions of DNA bases each cell division cycle. However, it can make errors, especially when the template DNA is damaged. In these cases, replication-coupled repair mechanisms remove the mistake or repair the template lesions to ensure high fidelity and complete copying of the genome. Failures in these genome maintenance activities generate mutations, rearrangements, and chromosome segregation problems that cause many human diseases. In this review, I provide a broad overview of replication-coupled repair pathways, explaining how they fix polymerase mistakes, respond to template damage that acts as obstacles to the replisome, deal with broken forks, and impact human health and disease.


Assuntos
Reparo do DNA/genética , Replicação do DNA/genética , Doenças Genéticas Inatas/genética , Genoma Humano/genética , Ciclo Celular/genética , Segregação de Cromossomos/genética , Dano ao DNA/genética , Instabilidade Genômica/genética , Humanos , Mutação/genética
7.
Nat Commun ; 10(1): 2588, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197172

RESUMO

The brain is a genomic mosaic shaped by cellular responses to genome damage. Here, we manipulate somatic genome stability by conditional Knl1 deletion from embryonic mouse brain. KNL1 mutations cause microcephaly and KNL1 mediates the spindle assembly checkpoint, a safeguard against chromosome missegregation and aneuploidy. We find that following Knl1 deletion, segregation errors in mitotic neural progenitor cells give rise to DNA damage on the missegregated chromosomes. This triggers rapid p53 activation and robust apoptotic and microglial phagocytic responses that extensively eliminate cells with somatic genome damage, thus causing microcephaly. By leaving only karyotypically normal progenitors to continue dividing, these mechanisms provide a second safeguard against brain somatic aneuploidy. Without Knl1 or p53-dependent safeguards, genome-damaged cells are not cleared, alleviating microcephaly, but paradoxically leading to total pre-weaning lethality. Thus, mitotic genome damage activates robust responses to eliminate somatic mutant cells, which if left unpurged, can impact brain and organismal fitness.


Assuntos
Aneuploidia , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neurais/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/genética , Segregação de Cromossomos/genética , Dano ao DNA/genética , Modelos Animais de Doenças , Embrião de Mamíferos , Instabilidade Genômica , Humanos , Cinetocoros/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Cultura Primária de Células , Deleção de Sequência , Fuso Acromático/metabolismo
8.
Nat Commun ; 10(1): 2354, 2019 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-31142748

RESUMO

In allopolyploids, correct chromosome segregation requires suppression of non-homologous crossovers while levels of homologous crossovers are ensured. To date, no mechanism able to specifically inhibit non-homologous crossovers has been described in allopolyploids other than in bread wheat. Here, we show that reducing the number of functional copies of MSH4, an essential gene for the main crossover pathway, prevents non-homologous crossovers in allotetraploid Brassica napus. We show that non-homologous crossovers originate almost exclusively from the MSH4-dependent recombination pathway and that their numbers decrease when MSH4 returns to single copy in B. napus; by contrast, homologous crossovers remain unaffected by MSH4 duplicate loss. We also demonstrate that MSH4 systematically returns to single copy following numerous independent polyploidy events, a pattern that is probably not by chance. These results suggest that stabilization of allopolyploid meiosis can be enhanced by loss of a key meiotic recombination gene.


Assuntos
Brassica napus/genética , Segregação de Cromossomos/genética , Troca Genética/genética , Meiose/genética , Proteínas MutS/genética , Poliploidia , Cromossomos de Plantas/metabolismo , Variações do Número de Cópias de DNA , Recombinação Homóloga
9.
Nat Genet ; 51(5): 824-834, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31036964

RESUMO

Chromosome segregation errors cause aneuploidy and genomic heterogeneity, which are hallmarks of cancer in humans. A persistent high frequency of these errors (chromosomal instability (CIN)) is predicted to profoundly impact tumor evolution and therapy response. It is unknown, however, how prevalent CIN is in human tumors. Using three-dimensional live-cell imaging of patient-derived tumor organoids (tumor PDOs), we show that CIN is widespread in colorectal carcinomas regardless of background genetic alterations, including microsatellite instability. Cell-fate tracking showed that, although mitotic errors are frequently followed by cell death, some tumor PDOs are largely insensitive to mitotic errors. Single-cell karyotype sequencing confirmed heterogeneity of copy number alterations in tumor PDOs and showed that monoclonal lines evolved novel karyotypes over time in vitro. We conclude that ongoing CIN is common in colorectal cancer organoids, and propose that CIN levels and the tolerance for mitotic errors shape aneuploidy landscapes and karyotype heterogeneity.


Assuntos
Instabilidade Cromossômica , Neoplasias Colorretais/genética , Aneuploidia , Linhagem Celular Tumoral , Segregação de Cromossomos , Neoplasias Colorretais/patologia , Variações do Número de Cópias de DNA , Humanos , Imagem Tridimensional , Cariótipo , Cariotipagem , Instabilidade de Microssatélites , Mitose/genética , Mutação , Organoides/patologia , Análise de Célula Única
10.
Mol Cell ; 74(5): 1069-1085.e11, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31000436

RESUMO

Orderly segregation of chromosomes during meiosis requires that crossovers form between homologous chromosomes by recombination. Programmed DNA double-strand breaks (DSBs) initiate meiotic recombination. We identify ANKRD31 as a key component of complexes of DSB-promoting proteins that assemble on meiotic chromosome axes. Genome-wide, ANKRD31 deficiency causes delayed recombination initiation. In addition, loss of ANKRD31 alters DSB distribution because of reduced selectivity for sites that normally attract DSBs. Strikingly, ANKRD31 deficiency also abolishes uniquely high rates of recombination that normally characterize pseudoautosomal regions (PARs) of X and Y chromosomes. Consequently, sex chromosomes do not form crossovers, leading to chromosome segregation failure in ANKRD31-deficient spermatocytes. These defects co-occur with a genome-wide delay in assembling DSB-promoting proteins on autosome axes and loss of a specialized PAR-axis domain that is highly enriched for DSB-promoting proteins in wild type. Thus, we propose a model for spatiotemporal patterning of recombination by ANKRD31-dependent control of axis-associated DSB-promoting proteins.


Assuntos
Proteínas de Transporte/genética , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga/genética , Meiose/genética , Animais , Proteínas de Transporte/química , Segregação de Cromossomos/genética , Masculino , Camundongos , Regiões Pseudoautossômicas/genética , Espermatócitos/crescimento & desenvolvimento , Espermatócitos/metabolismo , Cromossomo X/genética , Cromossomo Y/genética
11.
Mol Cell ; 74(5): 1053-1068.e8, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31003867

RESUMO

Double-strand breaks (DSBs) initiate the homologous recombination that is crucial for meiotic chromosome pairing and segregation. Here, we unveil mouse ANKRD31 as a lynchpin governing multiple aspects of DSB formation. Spermatocytes lacking ANKRD31 have altered DSB locations and fail to target DSBs to the pseudoautosomal regions (PARs) of sex chromosomes. They also have delayed and/or fewer recombination sites but, paradoxically, more DSBs, suggesting DSB dysregulation. Unrepaired DSBs and pairing failures-stochastic on autosomes, nearly absolute on X and Y-cause meiotic arrest and sterility in males. Ankrd31-deficient females have reduced oocyte reserves. A crystal structure defines a pleckstrin homology (PH) domain in REC114 and its direct intermolecular contacts with ANKRD31. In vivo, ANKRD31 stabilizes REC114 association with the PAR and elsewhere. Our findings inform a model in which ANKRD31 is a scaffold anchoring REC114 and other factors to specific genomic locations, thereby regulating DSB formation.


Assuntos
Recombinação Homóloga/genética , Meiose/genética , Recombinases/química , Animais , Pareamento Cromossômico , Segregação de Cromossomos/genética , Cromossomos , Cristalografia por Raios X , Quebras de DNA de Cadeia Dupla , Feminino , Masculino , Camundongos , Conformação Proteica , Recombinases/genética , Espermatócitos/química , Espermatócitos/metabolismo
12.
Nat Commun ; 10(1): 1673, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975984

RESUMO

Accurate chromosome segregation relies on microtubule end conversion, the ill-understood ability of kinetochores to transit from lateral microtubule attachment to durable association with dynamic microtubule plus-ends. The molecular requirements for this conversion and the underlying biophysical mechanisms are elusive. We reconstituted end conversion in vitro using two kinetochore components: the plus end-directed kinesin CENP-E and microtubule-binding Ndc80 complex, combined on the surface of a microbead. The primary role of CENP-E is to ensure close proximity between Ndc80 complexes and the microtubule plus-end, whereas Ndc80 complexes provide lasting microtubule association by diffusing on the microtubule wall near its tip. Together, these proteins mediate robust plus-end coupling during several rounds of microtubule dynamics, in the absence of any specialized tip-binding or regulatory proteins. Using a Brownian dynamics model, we show that end conversion is an emergent property of multimolecular ensembles of microtubule wall-binding proteins with finely tuned force-dependent motility characteristics.


Assuntos
Segregação de Cromossomos , Cinesina/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Animais , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/isolamento & purificação , Proteínas Cromossômicas não Histona/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Dinâmica não Linear , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , Imagem Individual de Molécula , Processos Estocásticos , Proteínas de Xenopus/genética , Proteínas de Xenopus/isolamento & purificação , Proteínas de Xenopus/metabolismo
13.
Cell Mol Life Sci ; 76(11): 2217-2229, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30980108

RESUMO

As the female gamete, meiotic oocytes provide not only half of the genome but also almost all stores for fertilization and early embryonic development. Because de novo mRNA transcription is absent in oocyte meiosis, protein-level regulations, especially the ubiquitin proteasome system, are more crucial. As the largest family of ubiquitin E3 ligases, Skp1-Cullin-F-box complexes recognize their substrates via F-box proteins with substrate-selected specificity. However, the variety of F-box proteins and their unknown substrates hinder our understanding of their functions. In this report, we find that Fbxo30, a new member of F-box proteins, is enriched in mouse oocytes, and its expression level declines substantially after the metaphase of the first meiosis (MI). Notably, depletion of Fbxo30 causes significant chromosome compaction accompanied by chromosome segregation failure and arrest at the MI stage, and this arrest is not caused by over-activation of spindle assembly checkpoint. Using immunoprecipitation and mass spectrometric analysis, we identify stem-loop-binding protein (SLBP) as a novel substrate of Fbxo30. SLBP overexpression caused by Fbxo30 depletion results in a remarkable overload of histone H3 on chromosomes that excessively condenses chromosomes and inhibits chromosome segregation. Our finding uncovers an unidentified pathway-controlling chromosome segregation and cell progress.


Assuntos
Segregação de Cromossomos , Cromossomos de Mamíferos/metabolismo , Proteínas F-Box/genética , Histonas/genética , Meiose , Proteínas Nucleares/genética , Oócitos/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Animais , Cromossomos de Mamíferos/ultraestrutura , Proteínas F-Box/antagonistas & inibidores , Proteínas F-Box/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Proteínas Nucleares/metabolismo , Oócitos/ultraestrutura , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
14.
DNA Cell Biol ; 38(6): 532-540, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30985224

RESUMO

Anillin is an actin binding protein and plays crucial roles during mitotic cell cycle progression in metazoan. However, the sequence and functions of the Anillin gene have not been yet characterized in the silkworm, Bombyx mori. In this study, we cloned the full-length cDNA sequence of the silkworm Anillin (BmAnillin) gene. The deduced amino acid sequence for BmAnillin protein comprises an Anillin homology region (AHR) covering an Anillin homology domain and a pleckstrin homology domain. Phylogenetic analysis and multiple alignments of the Anillin genes from silkworm and other organisms indicated evolutionary conservation in the AHR containing conserved phosphorylation sites. Reverse transcription-PCR experiments confirmed that the BmAnillin gene was highly expressed during larval development of gonads in which cells undergo mitotic cycles and exhibited an unexpected high expression in silk gland with endocycle during larval molting. RNA interference-mediated knockdown of the BmAnillin gene in silkworm BmN4-SID1 cells derived from ovary disrupted chromosome separation and resulted in a loss of the F-actin filament at cleavage furrow during anaphase, suggesting that the BmAnillin gene is essential for cytokinesis in silkworm.


Assuntos
Bombyx/genética , Proteínas Contráteis/genética , Animais , Bombyx/metabolismo , Células Cultivadas , Segregação de Cromossomos , Clonagem Molecular , Proteínas Contráteis/metabolismo , Proteínas Contráteis/fisiologia , Citocinese , Expressão Gênica , Genes de Insetos , Filogenia , Alinhamento de Sequência , Análise de Sequência
15.
Nat Commun ; 10(1): 1761, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30988289

RESUMO

During mitosis, tension develops across the centromere as a result of spindle-based forces. Metaphase tension may be critical in preventing mitotic chromosome segregation errors, however, the nature of force transmission at the centromere and the role of centromere mechanics in controlling metaphase tension remains unknown. We combined quantitative, biophysical microscopy with computational analysis to elucidate the mechanics of the centromere in unperturbed, mitotic human cells. We discovered that the mechanical stiffness of the human centromere matures during mitotic progression, which leads to amplified centromere tension specifically at metaphase. Centromere mechanical maturation is disrupted across multiple aneuploid cell lines, leading to a weak metaphase tension signal. Further, increasing deficiencies in centromere mechanical maturation are correlated with rising frequencies of lagging, merotelic chromosomes in anaphase, leading to segregation defects at telophase. Thus, we reveal a centromere maturation process that may be critical to the fidelity of chromosome segregation during mitosis.


Assuntos
Centrômero/fisiologia , Segregação de Cromossomos/fisiologia , Mitose/fisiologia , Aneuploidia , Linhagem Celular Tumoral , Células HeLa , Humanos , Metáfase , Modelos Biológicos , Fuso Acromático
16.
BMC Plant Biol ; 19(1): 157, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31023214

RESUMO

BACKGROUND: Pedicel orientation can affect the female flower orientation and seed yield in cucumber. A spontaneous mutant possessing upward growth of pedicels was identified in the wild type inbred strain 9930 and named upward-pedicel (up). The morphological and genetic analyses of up were performed in this study. In order to clone the up gene, 933 F2 individuals and 524 BC1 individuals derived from C-8-6 (WT) and up were used for map-based cloning. RESULTS: up was mapped to a 35.2 kb physical interval on chromosome 1, which contains three predicted genes. Sequencing analysis revealed that a 5-bp deletion was found in the second exon of Csa1G535800, and it led to a frameshift mutation resulting in a premature stop codon. The candidate gene of CsUp (Csa1G535800) was further confirmed via genomic and cDNA sequencing in biparental and natural cucumber populations. Sequencing data showed that a 4-bp deletion was found in the sixth exon of Csa1G535800 in CGN19839, another inbred line, and there was also a mutation of an amino acid in Csa1G535800 that could contribute to the upward growth of pedicels in CGN19839. Moreover, it was found that Csa1G535800 exhibited strong expression in the pedicel of WT, suggesting its important role in development of pedicel orientation. Thus, Csa1G535800 was considered to be the candidate gene of CsUp. CONCLUSIONS: CsUp encodes an Auxilin-like protein and controls pedicel orientation in cucumber. The identification of CsUp may help us to understand the mechanism of pedicel orientation development and allow for investigation of novel functions of Auxilin-like proteins in cucumber.


Assuntos
Auxilinas/genética , Mapeamento Cromossômico , Cucumis sativus/genética , Genes de Plantas , Estudos de Associação Genética , Mutação/genética , Sequência de Aminoácidos , Sequência de Bases , Segregação de Cromossomos , Cromossomos de Plantas/genética , Cruzamentos Genéticos , Regulação da Expressão Gênica de Plantas , Genes Recessivos , Loci Gênicos , Fenótipo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Característica Quantitativa Herdável , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
17.
PLoS Genet ; 15(3): e1008004, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30921322

RESUMO

Germ cell immortality, or transgenerational maintenance of the germ line, could be promoted by mechanisms that could occur in either mitotic or meiotic germ cells. Here we report for the first time that the GSP-2 PP1/Glc7 phosphatase promotes germ cell immortality. Small RNA-induced genome silencing is known to promote germ cell immortality, and we identified a separation-of-function allele of C. elegans gsp-2 that is compromised for germ cell immortality and is also defective for small RNA-induced genome silencing and meiotic but not mitotic chromosome segregation. Previous work has shown that GSP-2 is recruited to meiotic chromosomes by LAB-1, which also promoted germ cell immortality. At the generation of sterility, gsp-2 and lab-1 mutant adults displayed germline degeneration, univalents, histone methylation and histone phosphorylation defects in oocytes, phenotypes that mirror those observed in sterile small RNA-mediated genome silencing mutants. Our data suggest that a meiosis-specific function of GSP-2 ties small RNA-mediated silencing of the epigenome to germ cell immortality. We also show that transgenerational epigenomic silencing at hemizygous genetic elements requires the GSP-2 phosphatase, suggesting a functional link to small RNAs. Given that LAB-1 localizes to the interface between homologous chromosomes during pachytene, we hypothesize that small localized discontinuities at this interface could promote genomic silencing in a manner that depends on small RNAs and the GSP-2 phosphatase.


Assuntos
Células Germinativas/metabolismo , Proteína Fosfatase 1/fisiologia , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Genoma , Células Germinativas/fisiologia , Meiose/fisiologia , Prófase Meiótica I/fisiologia , Metilação , Monoéster Fosfórico Hidrolases , Proteína Fosfatase 1/metabolismo , Interferência de RNA/fisiologia , RNA Interferente Pequeno
18.
Mol Cell ; 74(4): 664-673.e5, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-30922844

RESUMO

Cohesin is a conserved, ring-shaped protein complex that topologically embraces DNA. Its central role in genome organization includes functions in sister chromatid cohesion, DNA repair, and transcriptional regulation. Cohesin loading onto chromosomes requires the Scc2-Scc4 cohesin loader, whose presence on chromatin in budding yeast depends on the RSC chromatin remodeling complex. Here we reveal a dual role of RSC in cohesin loading. RSC acts as a chromatin receptor that recruits Scc2-Scc4 by a direct protein interaction independent of chromatin remodeling. In addition, chromatin remodeling is required to generate a nucleosome-free region that is the substrate for cohesin loading. An engineered cohesin loading module can be created by fusing the Scc2 C terminus to RSC or to other chromatin remodelers, but not to unrelated DNA binding proteins. These observations demonstrate the importance of nucleosome-free DNA for cohesin loading and provide insight into how cohesin accesses DNA during its varied chromosomal activities.


Assuntos
Proteínas de Ciclo Celular/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Saccharomyces cerevisiae/genética , Segregação de Cromossomos/genética , Cromossomos/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Nucleossomos/genética , Saccharomyces cerevisiae/genética , Troca de Cromátide Irmã , Transcrição Genética
19.
Nat Commun ; 10(1): 981, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30816115

RESUMO

Animal cells undergo rapid rounding during mitosis, ensuring proper chromosome segregation, during which an outward rounding force abruptly increases upon prometaphase entry and is maintained at a constant level during metaphase. Initial cortical tension is generated by the actomyosin system to which both myosin motors and actin network architecture contribute. However, how cortical tension is maintained and its physiological significance remain unknown. We demonstrate here that Cdk1-mediated phosphorylation of DIAPH1 stably maintains cortical tension after rounding and inactivates the spindle assembly checkpoint (SAC). Cdk1 phosphorylates DIAPH1, preventing profilin1 binding to maintain cortical tension. Mutation of DIAPH1 phosphorylation sites promotes cortical F-actin accumulation, increases cortical tension, and delays anaphase onset due to SAC activation. Measurement of the intra-kinetochore length suggests that Cdk1-mediated cortex relaxation is indispensable for kinetochore stretching. We thus uncovered a previously unknown mechanism by which Cdk1 coordinates cortical tension maintenance and SAC inactivation at anaphase onset.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Quinase CDC2/metabolismo , Segregação de Cromossomos/fisiologia , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Anáfase/fisiologia , Ciclina B1/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Cinetocoros/metabolismo , Metáfase/fisiologia , Fosforilação , Profilinas/química , Profilinas/genética , Profilinas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
20.
PLoS Negl Trop Dis ; 13(3): e0007256, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30897087

RESUMO

Aurora kinases constitute a family of enzymes that play a key role during metazoan cells division, being involved in events like centrosome maturation and division, chromatin condensation, mitotic spindle assembly, control of kinetochore-microtubule attachments, and cytokinesis initiation. In this work, three Aurora kinase homologues were identified in Trypanosoma cruzi (TcAUK1, -2 and -3), a protozoan parasite of the Kinetoplastida Class. The genomic organization of these enzymes was fully analyzed, demonstrating that TcAUK1 is a single-copy gene, TcAUK2 coding sequence is present in two different forms (short and long) and TcAUK3 is a multi-copy gene. The three TcAUK genes are actively expressed in the different life cycle forms of T. cruzi (amastigotes, trypomastigotes and epimastigotes). TcAUK1 showed a changing localization along the cell cycle of the proliferating epimastigote form: at interphase it is located at the extremes of the kinetoplast while in mitosis it is detected at the cell nucleus, in close association with the mitotic spindle. Overexpression of TcAUK1 in epimastigotes leaded to a delay in the G2/M phases of the cell cycle due a retarded beginning of kinetoplast duplication. By immunofluorescence, we found that when it was overexpressed TcAUK1 lost its localization at the extremes of the kinetoplast during interphase, being observed inside the cell nucleus throughout the entire cell cycle. In summary, TcAUK1 appears to be a functional homologue of human Aurora B kinase, as it is related to mitotic spindle assembling and chromosome segregation. Moreover, TcAUK1 also seems to play a role during the initiation of kinetoplast duplication, a novel role described for this protein.


Assuntos
Aurora Quinases/metabolismo , Estágios do Ciclo de Vida , Mitocôndrias/fisiologia , Trypanosoma cruzi/enzimologia , Aurora Quinases/genética , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Segregação de Cromossomos , Citocinese , Humanos , Mitose , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Fuso Acromático/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/fisiologia
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