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1.
Nature ; 582(7810): 119-123, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32494069

RESUMO

The three-dimensional architecture of the genome governs its maintenance, expression and transmission. The cohesin protein complex organizes the genome by topologically linking distant loci, and is highly enriched in specialized chromosomal domains surrounding centromeres, called pericentromeres1-6. Here we report the three-dimensional structure of pericentromeres in budding yeast (Saccharomyces cerevisiae) and establish the relationship between genome organization and function. We find that convergent genes mark pericentromere borders and, together with core centromeres, define their structure and function by positioning cohesin. Centromeres load cohesin, and convergent genes at pericentromere borders trap it. Each side of the pericentromere is organized into a looped conformation, with border convergent genes at the base. Microtubule attachment extends a single pericentromere loop, size-limited by convergent genes at its borders. Reorienting genes at borders into a tandem configuration repositions cohesin, enlarges the pericentromere and impairs chromosome biorientation during mitosis. Thus, the linear arrangement of transcriptional units together with targeted cohesin loading shapes pericentromeres into a structure that is competent for chromosome segregation. Our results reveal the architecture of the chromosomal region within which kinetochores are embedded, as well as the restructuring caused by microtubule attachment. Furthermore, we establish a direct, causal relationship between the three-dimensional genome organization of a specific chromosomal domain and cellular function.


Assuntos
Centrômero/genética , Centrômero/metabolismo , Genes Fúngicos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/metabolismo , Centrômero/química , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Genoma Fúngico/genética , Viabilidade Microbiana/genética , Mitose/genética , Conformação Molecular
2.
PLoS Biol ; 18(6): e3000718, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32516305

RESUMO

Autophagy is an intracellular degradation pathway targeting organelles and macromolecules, thereby regulating various cellular functions. Phosphorylation is a key posttranscriptional protein modification implicated in the regulation of biological function including autophagy. Under asynchronous conditions, autophagy activity is predominantly suppressed by mechanistic target of rapamycin (mTOR) kinase, but whether autophagy-related genes (ATG) proteins are phosphorylated differentially throughout the sequential phases of the cell cycle remains unclear. In this issue, Li and colleagues report that cyclin-dependent kinase 1 (CDK1) phosphorylates the ULK complex during mitosis. This phosphorylation induces autophagy and, surprisingly, is shown to drive cell cycle progression. This work reveals a yet-unappreciated role for autophagy in cell cycle progression and enhances our understanding of the specific phase-dependent autophagy regulation during cellular growth and proliferation.


Assuntos
Autofagia , Proteína Quinase CDC2 , Alvo Mecanístico do Complexo 1 de Rapamicina , Mitose , Fosforilação , Serina-Treonina Quinases TOR
3.
PLoS Biol ; 18(6): e3000288, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32516310

RESUMO

Unc-51-like autophagy activating kinase 1 (ULK1)-autophagy-related 13 (ATG13) is the most upstream autophagy initiation complex that is phosphorylated by mammalian target-of-rapamycin complex 1 (mTORC1) and AMP-activated protein kinase (AMPK) to induce autophagy in asynchronous conditions. However, their phospho-regulation and functions in mitosis and cell cycle remain unknown. Here we show that ULK1-ATG13 complex is differentially regulated throughout the cell cycle, especially in mitosis, in which both ULK1 and ATG13 are highly phosphorylated by the key cell cycle machinery cyclin-dependent kinase 1 (CDK1)/cyclin B. Combining mass spectrometry and site-directed mutagenesis, we found that CDK1-induced ULK1-ATG13 phosphorylation promotes mitotic autophagy and cell cycle progression. Moreover, double knockout (DKO) of ULK1 and ATG13 could block cell cycle progression and significantly decrease cancer cell proliferation in cell line and mouse models. Our results not only bridge the mutual regulation between the core machinery of autophagy and mitosis but also illustrate the positive function of ULK1-ATG13 and their phosphorylation by CDK1 in mitotic autophagy regulation.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Proteína Quinase CDC2/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitose , Animais , Anticorpos/metabolismo , Linhagem Celular , Ciclina B/metabolismo , Feminino , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação , Especificidade por Substrato
4.
PLoS Biol ; 18(6): e3000679, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32555591

RESUMO

Centriolar satellites are dynamic, membraneless granules composed of over 200 proteins. They store, modify, and traffic centrosome and primary cilium proteins, and help to regulate both the biogenesis and some functions of centrosomes and cilium. In most cell types, satellites cluster around the perinuclear centrosome, but their integrity and cellular distribution are dynamically remodeled in response to different stimuli, such as cell cycle cues. Dissecting the specific and temporal functions and mechanisms of satellites and how these are influenced by their cellular positioning and dynamics has been challenging using genetic approaches, particularly in ciliated and proliferating cells. To address this, we developed a chemical-based trafficking assay to rapidly and efficiently redistribute satellites to either the cell periphery or center, and fuse them into stable clusters in a temporally controlled way. Induced satellite clustering at either the periphery or center resulted in antagonistic changes in the pericentrosomal levels of a subset of proteins, revealing a direct and selective role for their positioning in protein targeting and sequestration. Systematic analysis of the interactome of peripheral satellite clusters revealed enrichment of proteins implicated in cilium biogenesis and mitosis. Importantly, induction of peripheral satellite targeting in ciliated cells revealed a function for satellites not just for efficient cilium assembly but also in the maintenance of steady-state cilia and in cilia disassembly by regulating the structural integrity of the ciliary axoneme. Finally, perturbing satellite distribution and dynamics inhibited their mitotic dissolution, and mitotic progression was perturbed only in cells with centrosomal satellite clustering. Collectively, our results for the first time showed a direct link between satellite functions and their pericentrosomal clustering, suggested new mechanisms underlying satellite functions during cilium assembly, and provided a new tool for probing temporal satellite functions in different contexts.


Assuntos
Centríolos/metabolismo , Cílios/metabolismo , Grânulos Citoplasmáticos/metabolismo , Autoantígenos/química , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Mitose , Fenótipo , Domínios Proteicos , Multimerização Proteica , Reprodutibilidade dos Testes
5.
Cancer Invest ; 38(5): 300-309, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32378982

RESUMO

Centrosome amplification leads to aberrant mitosis, giving rise to aneuploidy and it has been associated with poor prognosis in human cancers. This study aimed to evaluate the relationship between polyploidy, centrosome abnormalities, and response to endocrine treatment in progestin-induced mouse mammary carcinomas. We found cells with three or more centrosomes in the polyploid tumors. The endocrine unresponsive tumors showed a higher average number of centrosomes per cell than the responsive tumors. The results suggest an association between polyploidy and centrosome amplification with the resistance to endocrine therapy in this luminal breast cancer model.


Assuntos
Neoplasias da Mama/patologia , Carcinoma/patologia , Centrossomo/patologia , Hormônios/metabolismo , Aneuploidia , Animais , Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Linhagem Celular Tumoral , Centrossomo/metabolismo , Feminino , Amplificação de Genes/fisiologia , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Mitose/fisiologia , Poliploidia
6.
PLoS Comput Biol ; 16(4): e1007733, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251461

RESUMO

The cellular protein-protein interaction network that governs cellular proliferation (cell cycle) is highly complex. Here, we have developed a novel computational model of human mitotic cell cycle, integrating diverse cellular mechanisms, for the purpose of generating new hypotheses and predicting new experiments designed to help understand complex diseases. The pathogenic state investigated is infection by a human herpesvirus. The model starts at mitotic entry initiated by the activities of Cyclin-dependent kinase 1 (CDK1) and Polo-like kinase 1 (PLK1), transitions through Anaphase-promoting complex (APC/C) bound to Cell division cycle protein 20 (CDC20), and ends upon mitotic exit mediated by APC/C bound to CDC20 homolog 1 (CDH1). It includes syntheses and multiple mechanisms of degradations of the mitotic proteins. Prior to this work, no such comprehensive model of the human mitotic cell cycle existed. The new model is based on a hybrid framework combining Michaelis-Menten and mass action kinetics for the mitotic interacting reactions. It simulates temporal changes in 12 different mitotic proteins and associated protein complexes in multiple states using 15 interacting reactions and 26 ordinary differential equations. We have defined model parameter values using both quantitative and qualitative data and using parameter values from relevant published models, and we have tested the model to reproduce the cardinal features of human mitosis determined experimentally by numerous laboratories. Like cancer, viruses create dysfunction to support infection. By simulating infection of the human herpesvirus, cytomegalovirus, we hypothesize that virus-mediated disruption of APC/C is necessary to establish a unique mitotic collapse with sustained CDK1 activity, consistent with known mechanisms of virus egress. With the rapid discovery of cellular protein-protein interaction networks and regulatory mechanisms, we anticipate that this model will be highly valuable in helping us to understand the network dynamics and identify potential points of therapeutic interventions.


Assuntos
Biologia Computacional/métodos , Mitose/fisiologia , Mapas de Interação de Proteínas/fisiologia , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Antígenos CD/metabolismo , Proteína Quinase CDC2/metabolismo , Caderinas/metabolismo , Proteínas Cdc20/metabolismo , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Humanos , Cinética , Modelos Teóricos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo
7.
PLoS One ; 15(4): e0231665, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32287321

RESUMO

BACKGROUND: Many genomic analyses of cortisol-producing adrenocortical carcinoma (ACC) have been reported, but very few have come from East Asia. The first objective of this study is to verify the genetic difference with the previous reports by analyzing targeted deep sequencing of 7 Japanese ACC cases using next-generation sequencing (NGS). The second objective is to compare the somatic variant findings identified by NGS analysis with clinical and pathological findings, aiming to acquire new knowledge about the factors that contribute to the poor prognosis of ACC and to find new targets for the treatment of ACC. METHOD: DNA was extracted from ACC tissue of seven patients and two reference blood samples. Targeted deep sequencing was performed using the MiSeq system for 12 genes, and the obtained results were analyzed using MuTect2. The hypothesis was obtained by integrating the somatic variant findings with clinical and pathological data, and it was further verified using The Cancer Genome Atlas (TCGA) dataset for ACC. RESULTS: Six possible pathogenic and one uncertain significance somatic variants including a novel PRKAR1A (NM_002734.4):c.545C>A (p.T182K) variant were found in five of seven cases. By integrating these data with pathological findings, we hypothesized that cases with TP53 variants were more likely to show atypical mitotic figures. Using TCGA dataset, we found that atypical mitotic figures were associated with TP53 somatic variant, and mRNA expression of CCNB2 and AURKA was significantly high in TP53 mutated cases and atypical mitotic figure cases. CONCLUSION: We believe this is the first report that discusses the relationship between atypical mitotic figures and TP53 somatic variant in ACC. We presumed that overexpression of CCNB2 and AURKA mRNA may cause atypical mitosis in TP53 somatic mutated cases. Because AURKA is highly expressed in atypical mitotic cases, it may be an appropriate indicator for AURKA inhibitors.


Assuntos
Neoplasias do Córtex Suprarrenal/genética , Carcinoma Adrenocortical/genética , Aurora Quinase A/genética , Ciclina B2/genética , Mitose , Regulação para Cima , Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/patologia , Adulto , Aurora Quinase A/metabolismo , Ciclina B2/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/genética
8.
Science ; 368(6488)2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32299917

RESUMO

The chromosome breakage-fusion-bridge (BFB) cycle is a mutational process that produces gene amplification and genome instability. Signatures of BFB cycles can be observed in cancer genomes alongside chromothripsis, another catastrophic mutational phenomenon. We explain this association by elucidating a mutational cascade that is triggered by a single cell division error-chromosome bridge formation-that rapidly increases genomic complexity. We show that actomyosin forces are required for initial bridge breakage. Chromothripsis accumulates, beginning with aberrant interphase replication of bridge DNA. A subsequent burst of DNA replication in the next mitosis generates extensive DNA damage. During this second cell division, broken bridge chromosomes frequently missegregate and form micronuclei, promoting additional chromothripsis. We propose that iterations of this mutational cascade generate the continuing evolution and subclonal heterogeneity characteristic of many human cancers.


Assuntos
Carcinogênese/genética , Carcinogênese/patologia , Quebra Cromossômica , Dano ao DNA/genética , Mitose/genética , Neoplasias/genética , Neoplasias/patologia , Actomiosina/metabolismo , Linhagem Celular Tumoral , Exodesoxirribonucleases/genética , Dosagem de Genes , Genoma Humano , Humanos , Fenômenos Mecânicos , Mutagênese , Mutação , Fosfoproteínas/genética , Análise de Célula Única
9.
Adv Exp Med Biol ; 1243: 21-40, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32297209

RESUMO

The cell cycle is a sophisticated space-time regulated mechanism where a wide variety of protein modules and complexes associate functioning in a concerted manner to regulate and transfer the genetic material to daughter cells. CCT (chaperonin containing TCP-1, also known as TRiC) is a molecular machine that forms a high molecular weight complex (1000 KDa). CCT is emerging as a key molecule during mitosis due to its essential role in the folding of many important proteins involved in cell division (Cdh1, Plk1, p27, Cdc20, PP2a regulatory subunits, tubulin or actin) suggesting its involvement in uncontrolled proliferation. The assembly is formed by eight different subunits called CCTα, ß, γ, δ, ε, ζ, η and θ in mammals corresponding to CCT1-8 in yeast. CCT/TRiC is organized in a unique intra- and inter-ring arrangement. The chaperonin monomers share a common domain structure including an equatorial domain, which contains all the inter-ring contacts, most of the intra-ring contacts and the ATP binding site, whose binding and hydrolysis triggers the conformational changes that take place during the functional cycle. All chaperonins display an open substrate-receptive conformation, where the unfolded protein is recognized and trapped, and a closed conformation where the substrate is isolated from the bulk of the intracellular environment. In this chapter we discuss the complex set of intra- and inter-ring allosteric signals during chaperonin function.


Assuntos
Proliferação de Células , Chaperonina com TCP-1/metabolismo , Regulação Alostérica , Animais , Chaperonina com TCP-1/química , Humanos , Mitose , Dobramento de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
10.
Nature ; 580(7804): 542-547, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32322059

RESUMO

Prolonged mitosis often results in apoptosis1. Shortened mitosis causes tumorigenic aneuploidy, but it is unclear whether it also activates the apoptotic machinery2. Separase, a cysteine protease and trigger of all eukaryotic anaphases, has a caspase-like catalytic domain but has not previously been associated with cell death3,4. Here we show that human cells that enter mitosis with already active separase rapidly undergo death in mitosis owing to direct cleavage of anti-apoptotic MCL1 and BCL-XL by separase. Cleavage not only prevents MCL1 and BCL-XL from sequestering pro-apoptotic BAK, but also converts them into active promoters of death in mitosis. Our data strongly suggest that the deadliest cleavage fragment, the C-terminal half of MCL1, forms BAK/BAX-like pores in the mitochondrial outer membrane. MCL1 and BCL-XL are turned into separase substrates only upon phosphorylation by NEK2A. Early mitotic degradation of this kinase is therefore crucial for preventing apoptosis upon scheduled activation of separase in metaphase. Speeding up mitosis by abrogation of the spindle assembly checkpoint results in a temporal overlap of the enzymatic activities of NEK2A and separase and consequently in cell death. We propose that NEK2A and separase jointly check on spindle assembly checkpoint integrity and eliminate cells that are prone to chromosome missegregation owing to accelerated progression through early mitosis.


Assuntos
Apoptose , Mitose , Separase/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Segregação de Cromossomos , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Camundongos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Quinases Relacionadas a NIMA/metabolismo , Fosforilação , Especificidade por Substrato , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína bcl-X/metabolismo
11.
Mutat Res ; 852: 503169, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32265043

RESUMO

The phycotoxins, okadaic acid (OA) and dinophysistoxins 1 and 2 (DTX-1 and -2), are protein phosphatase PP2A and PP1 inhibitors involved in diarrhetic shellfish poisoning (DSP) in humans. Data on the in vivo acute toxicity of the OA-group toxins show some differences and the European Food Safety Authority (EFSA) has determined toxicity equivalent factors (TEFs) of one for the reference toxin, OA, as well as for DTX-1 and 0.6 for DTX-2. However, recent in vitro studies indicated that DTX-1 seems to be more toxic than OA. As OA was described as apoptotic and aneugenic compound, we analyzed the DNA damage responses induced by the 3 toxins through γH2AX and pH3 biomarkers on proliferative HepaRG cells using High Content Analysis. We quantitatively examined the responses for γH2AX and pH3 by benchmark dose analyzing (BMD) using PROAST software. We found that the three toxins increased both γH2AX- and pH3-positive cells populations in a concentration-dependent manner. The 3 toxins induced mitotic arrest, characteristic of aneugenic compounds, as well as DNA strand-breaks concomitantly to cytotoxicity. BMD analysis showed that DTX-1 is the most potent inducer of DNA damage, followed by OA and DTX-2. The quantitative genotoxic data provided in this study are additional findings for reconsidering the estimated TEFs of this group of phycotoxins.


Assuntos
Inibidores Enzimáticos/toxicidade , Histonas/genética , Mutagênicos/toxicidade , Ácido Okadáico/toxicidade , Piranos/toxicidade , Benchmarking , Biomarcadores/metabolismo , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Dano ao DNA , Relação Dose-Resposta a Droga , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Histonas/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mitose/efeitos dos fármacos , Testes de Mutagenicidade , Fosforilação/efeitos dos fármacos , Software
12.
Mutat Res ; 850-851: 503147, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32247562

RESUMO

Bulbus Fritillariacirrhosa D. Don (BFC) has been widely used as an herbal medicament for respiratory diseases in China for over 2000 years. The ethnomedicinal effects of BFC have been scientifically verified, nevertheless its toxicity has not been completely studied. Previously, we have reported that the aqueous extract of BFC induces mitotic aberrations and chromosomal instability (CIN) in human colon epithelial NCM460 cells via dysfunctioning the mitotic checkpoint. Here, we extend this study and specifically focus on the influence of BFC on cytokinesis, the final step of cell division. One remarkable change in NCM460 cells following BFC treatment is the high incidence of binucleated cells (BNCs). More detailed investigation of the ana-telophases reveals that furrow ingression, the first stage of cytokinesis, is inhibited by BFC. Asynchronous cultures treatment demonstrates that furrow ingression defects induced by BFCs are highly associated with the formation of BNCs in ensuing interphase, indicating the BNCs phenotype after BFC treatment was resulted from cytokinesis failure. In line with this, the expression of genes involved in the regulation of furrow ingression is significantly de-regulated by BFC (e.g., LATS-1/2 and Aurora-B are upregulated, and YB-1 is downregulated). Furthermore, long-term treatment of BFC elucidates that the BNCs phenotype is transient and the loss of BNCs is associated with increased frequency of micronuclei and nuclear buds, two biomarkers of CIN. In supporting of these findings, the Nin Jiom Pei Pa Koa and Chuanbei Pipa Gao, two commercially available Chinese traditional medicines containing BFC, are able to induce multinucleation and CIN in NCM460 cells. Altogether, these data provide the first in vitro experimental evidence linking BFC to cytokinesis failure and suggest the resultant BNCs may be intermediates to produce CIN progenies.


Assuntos
Instabilidade Cromossômica/efeitos dos fármacos , Citocinese/efeitos dos fármacos , Fritillaria/química , Extratos Vegetais/farmacologia , Aurora Quinase B/genética , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Instabilidade Cromossômica/genética , Colo/efeitos dos fármacos , Colo/patologia , Citocinese/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mitose/efeitos dos fármacos , Extratos Vegetais/química , Raízes de Plantas/química , Proteínas Serina-Treonina Quinases/genética , Proteína 1 de Ligação a Y-Box/genética
13.
Proc Natl Acad Sci U S A ; 117(16): 8924-8933, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32265285

RESUMO

Adaptation to environmental changes is crucial for cell fitness. In Saccharomyces cerevisiae, variations in external osmolarity trigger the activation of the stress-activated protein kinase Hog1 (high-osmolarity glycerol 1), which regulates gene expression, metabolism, and cell-cycle progression. The activation of this kinase leads to the regulation of G1, S, and G2 phases of the cell cycle to prevent genome instability and promote cell survival. Here we show that Hog1 delays mitotic exit when cells are stressed during metaphase. Hog1 phosphorylates the nucleolar protein Net1, altering its affinity for the phosphatase Cdc14, whose activity is essential for mitotic exit and completion of the cell cycle. The untimely release of Cdc14 from the nucleolus upon activation of Hog1 is linked to a defect in ribosomal DNA (rDNA) and telomere segregation, and it ultimately delays cell division. A mutant of Net1 that cannot be phosphorylated by Hog1 displays reduced viability upon osmostress. Thus, Hog1 contributes to maximizing cell survival upon stress by regulating mitotic exit.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mitose/fisiologia , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , DNA Ribossômico/metabolismo , Mutação , Proteínas Nucleares/genética , Pressão Osmótica/fisiologia , Fosforilação/genética , Proteínas Tirosina Fosfatases/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Homeostase do Telômero/fisiologia
14.
PLoS Biol ; 18(4): e3000664, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32267835

RESUMO

The N6-methyladenosine (m6A) modification regulates mRNA stability and translation. Here, we show that transcriptomic m6A modification can be dynamic and the m6A reader protein YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) promotes mRNA decay during cell cycle. Depletion of YTHDF2 in HeLa cells leads to the delay of mitotic entry due to overaccumulation of negative regulators of cell cycle such as Wee1-like protein kinase (WEE1). We demonstrate that WEE1 transcripts contain m6A modification, which promotes their decay through YTHDF2. Moreover, we found that YTHDF2 protein stability is dependent on cyclin-dependent kinase 1 (CDK1) activity. Thus, CDK1, YTHDF2, and WEE1 form a feedforward regulatory loop to promote mitotic entry. We further identified Cullin 1 (CUL1), Cullin 4A (CUL4A), damaged DNA-binding protein 1 (DDB1), and S-phase kinase-associated protein 2 (SKP2) as components of E3 ubiquitin ligase complexes that mediate YTHDF2 proteolysis. Our study provides insights into how cell cycle mediators modulate transcriptomic m6A modification, which in turn regulates the cell cycle.


Assuntos
Adenosina/análogos & derivados , Ciclo Celular/fisiologia , Mitose/fisiologia , Proteínas de Ligação a RNA/metabolismo , Adenosina/genética , Adenosina/metabolismo , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Estabilidade Proteica , Proteínas Tirosina Quinases/genética , Estabilidade de RNA , Proteínas de Ligação a RNA/genética , Proteínas Quinases Associadas a Fase S/metabolismo
15.
Nat Commun ; 11(1): 1746, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32269212

RESUMO

In mammalian cell lines, the endosomal sorting complex required for transport (ESCRT)-III mediates abscission, the process that physically separates daughter cells and completes cell division. Cep55 protein is regarded as the master regulator of abscission, because it recruits ESCRT-III to the midbody (MB), the site of abscission. However, the importance of this mechanism in a mammalian organism has never been tested. Here we show that Cep55 is dispensable for mouse embryonic development and adult tissue homeostasis. Cep55-knockout offspring show microcephaly and primary neural progenitors require Cep55 and ESCRT for survival and abscission. However, Cep55 is dispensable for cell division in embryonic or adult tissues. In vitro, division of primary fibroblasts occurs without Cep55 and ESCRT-III at the midbody and is not affected by ESCRT depletion. Our work defines Cep55 as an abscission regulator only in specific tissue contexts and necessitates the re-evaluation of an alternative ESCRT-independent cell division mechanism.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Citocinese , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Animais , Animais Recém-Nascidos , Apoptose , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Células Cultivadas , Córtex Cerebral/anormalidades , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fibroblastos/metabolismo , Deleção de Genes , Genótipo , Rim/anormalidades , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microcefalia/patologia , Mitose
16.
PLoS Biol ; 18(4): e3000538, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32339165

RESUMO

Oogenesis is a complex developmental process that involves spatiotemporally regulated coordination between the germline and supporting, somatic cell populations. This process has been modeled extensively using the Drosophila ovary. Although different ovarian cell types have been identified through traditional means, the large-scale expression profiles underlying each cell type remain unknown. Using single-cell RNA sequencing technology, we have built a transcriptomic data set for the adult Drosophila ovary and connected tissues. Using this data set, we identified the transcriptional trajectory of the entire follicle-cell population over the course of their development from stem cells to the oogenesis-to-ovulation transition. We further identify expression patterns during essential developmental events that take place in somatic and germline cell types such as differentiation, cell-cycle switching, migration, symmetry breaking, nurse-cell engulfment, egg-shell formation, and corpus luteum signaling. Extensive experimental validation of unique expression patterns in both ovarian and nearby, nonovarian cells also led to the identification of many new cell type-and stage-specific markers. The inclusion of several nearby tissue types in this data set also led to our identification of functional convergence in expression between distantly related cell types such as the immune-related genes that were similarly expressed in immune cells (hemocytes) and ovarian somatic cells (stretched cells) during their brief phagocytic role in nurse-cell engulfment. Taken together, these findings provide new insight into the temporal regulation of genes in a cell-type specific manner during oogenesis and begin to reveal the relatedness in expression between cell and tissues types.


Assuntos
Drosophila melanogaster/citologia , Oogênese/genética , Ovário/citologia , Animais , Animais Geneticamente Modificados , Diferenciação Celular/genética , Linhagem da Célula , Drosophila melanogaster/genética , Feminino , Perfilação da Expressão Gênica , Marcadores Genéticos , Hemócitos/citologia , Hemócitos/fisiologia , Mitose/genética , Folículo Ovariano/citologia , Ovário/fisiologia , Ovulação/genética , Análise de Sequência de RNA , Análise de Célula Única/métodos
17.
Nat Commun ; 11(1): 1851, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32296040

RESUMO

Genome stability relies on proper coordination of mitosis and cytokinesis, where dynamic microtubules capture and faithfully segregate chromosomes into daughter cells. With a high-content RNAi imaging screen targeting more than 2,000 human lncRNAs, we identify numerous lncRNAs involved in key steps of cell division such as chromosome segregation, mitotic duration and cytokinesis. Here, we provide evidence that the chromatin-associated lncRNA, linc00899, leads to robust mitotic delay upon its depletion in multiple cell types. We perform transcriptome analysis of linc00899-depleted cells and identify the neuronal microtubule-binding protein, TPPP/p25, as a target of linc00899. We further show that linc00899 binds TPPP/p25 and suppresses its transcription. In cells depleted of linc00899, upregulation of TPPP/p25 alters microtubule dynamics and delays mitosis. Overall, our comprehensive screen uncovers several lncRNAs involved in genome stability and reveals a lncRNA that controls microtubule behaviour with functional implications beyond cell division.


Assuntos
Divisão Celular/genética , Divisão Celular/fisiologia , RNA Longo não Codificante/genética , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Mitose/genética , Mitose/fisiologia , Proteínas/genética , Interferência de RNA/fisiologia
18.
PLoS One ; 15(4): e0227849, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32343690

RESUMO

Understanding the mitotic DNA damage response (DDR) is critical to our comprehension of cancer, premature aging and developmental disorders which are marked by DNA repair deficiencies. In this study we use a micro-focused laser to induce DNA damage in selected mitotic chromosomes to study the subsequent repair response. Our findings demonstrate that (1) mitotic cells are capable of DNA repair as evidenced by DNA synthesis at damage sites, (2) Repair is attenuated when DNA-PKcs and ATM are simultaneously compromised, (3) Laser damage may permit the observation of previously undetected DDR proteins when damage is elicited by other methods in mitosis, and (4) Twenty five percent of mitotic DNA-damaged cells undergo a subsequent mitosis. Together these findings suggest that mitotic DDR is more complex than previously thought and may involve factors from multiple repair pathways that are better understood in interphase.


Assuntos
Quebras de DNA/efeitos da radiação , Reparo do DNA , DNA/biossíntese , Fase G1/genética , Mitose/genética , Animais , Linhagem Celular , DNA/genética , DNA/efeitos da radiação , Fase G1/efeitos da radiação , Humanos , Raios Infravermelhos/efeitos adversos , Lasers/efeitos adversos , Mitose/efeitos da radiação , Potoroidae
19.
Nat Commun ; 11(1): 2055, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345962

RESUMO

Breast cancer susceptibility gene II (BRCA2) is central in homologous recombination (HR). In meiosis, BRCA2 binds to MEILB2 to localize to DNA double-strand breaks (DSBs). Here, we identify BRCA2 and MEILB2-associating protein 1 (BRME1), which functions as a stabilizer of MEILB2 by binding to an α-helical N-terminus of MEILB2 and preventing MEILB2 self-association. BRCA2 binds to the C-terminus of MEILB2, resulting in the formation of the BRCA2-MEILB2-BRME1 ternary complex. In Brme1 knockout (Brme1-/-) mice, the BRCA2-MEILB2 complex is destabilized, leading to defects in DSB repair, homolog synapsis, and crossover formation. Persistent DSBs in Brme1-/- reactivate the somatic-like DNA-damage response, which repairs DSBs but cannot complement the crossover formation defects. Further, MEILB2-BRME1 is activated in many human cancers, and somatically expressed MEILB2-BRME1 impairs mitotic HR. Thus, the meiotic BRCA2 complex is central in meiotic HR, and its misregulation is implicated in cancer development.


Assuntos
Proteína BRCA2/metabolismo , Recombinação Homóloga/genética , Meiose/genética , Mitose/genética , Complexos Multiproteicos/metabolismo , Neoplasias/genética , Rad51 Recombinase/metabolismo , Alelos , Animais , Linhagem Celular Tumoral , Pareamento Cromossômico , Quebras de DNA de Cadeia Dupla , Masculino , Camundongos Endogâmicos C57BL , Ligação Proteica , Estabilidade Proteica , Espermatozoides/metabolismo
20.
Sheng Wu Gong Cheng Xue Bao ; 36(4): 612-621, 2020 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-32347056

RESUMO

Apomixis has been widely concerned because of its great potential in heterosis fixation. Artificial apomixis is an important direction of current apomixis research. Mitosis instead of Meiosis (MIME) produces diploid gametes that is identical with the maternal genetic composition and is a key step in the artificial creation of apomixes. This paper reviews the occurrence of MIME and its application in crop apomixis and the problems encountered, in an aim to provide reference for expanding the application of MIME in crop apomixis.


Assuntos
Apomixia , Produtos Agrícolas/genética , Meiose , Mitose , Diploide , Células Germinativas
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