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1.
Cell Syst ; 12(8): 780-794.e7, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34139154

RESUMO

COVID-19 is highly variable in its clinical presentation, ranging from asymptomatic infection to severe organ damage and death. We characterized the time-dependent progression of the disease in 139 COVID-19 inpatients by measuring 86 accredited diagnostic parameters, such as blood cell counts and enzyme activities, as well as untargeted plasma proteomes at 687 sampling points. We report an initial spike in a systemic inflammatory response, which is gradually alleviated and followed by a protein signature indicative of tissue repair, metabolic reconstitution, and immunomodulation. We identify prognostic marker signatures for devising risk-adapted treatment strategies and use machine learning to classify therapeutic needs. We show that the machine learning models based on the proteome are transferable to an independent cohort. Our study presents a map linking routinely used clinical diagnostic parameters to plasma proteomes and their dynamics in an infectious disease.


Assuntos
Biomarcadores/análise , COVID-19/patologia , Progressão da Doença , Proteoma/fisiologia , Fatores Etários , Contagem de Células Sanguíneas , Gasometria , Ativação Enzimática , Humanos , Inflamação/patologia , Aprendizado de Máquina , Prognóstico , Proteômica , SARS-CoV-2/imunologia
2.
Elife ; 92020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32543370

RESUMO

Microbial fitness screens are a key technique in functional genomics. We present an all-in-one solution, pyphe, for automating and improving data analysis pipelines associated with large-scale fitness screens, including image acquisition and quantification, data normalisation, and statistical analysis. Pyphe is versatile and processes fitness data from colony sizes, viability scores from phloxine B staining or colony growth curves, all obtained with inexpensive transilluminating flatbed scanners. We apply pyphe to show that the fitness information contained in late endpoint measurements of colony sizes is similar to maximum growth slopes from time series. We phenotype gene-deletion strains of fission yeast in 59,350 individual fitness assays in 70 conditions, revealing that colony size and viability provide complementary, independent information. Viability scores obtained from quantifying the redness of phloxine-stained colonies accurately reflect the fraction of live cells within colonies. Pyphe is user-friendly, open-source and fully documented, illustrated by applications to diverse fitness analysis scenarios.


Assuntos
Sobrevivência Celular , Contagem de Colônia Microbiana/métodos , Aptidão Genética , Fenótipo , Schizosaccharomyces/fisiologia , Contagem de Colônia Microbiana/instrumentação , Deleção de Genes , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Software
3.
Mol Syst Biol ; 16(4): e9270, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32319721

RESUMO

Cells balance glycolysis with respiration to support their metabolic needs in different environmental or physiological contexts. With abundant glucose, many cells prefer to grow by aerobic glycolysis or fermentation. Using 161 natural isolates of fission yeast, we investigated the genetic basis and phenotypic effects of the fermentation-respiration balance. The laboratory and a few other strains depended more on respiration. This trait was associated with a single nucleotide polymorphism in a conserved region of Pyk1, the sole pyruvate kinase in fission yeast. This variant reduced Pyk1 activity and glycolytic flux. Replacing the "low-activity" pyk1 allele in the laboratory strain with the "high-activity" allele was sufficient to increase fermentation and decrease respiration. This metabolic rebalancing triggered systems-level adjustments in the transcriptome and proteome and in cellular traits, including increased growth and chronological lifespan but decreased resistance to oxidative stress. Thus, low Pyk1 activity does not lead to a growth advantage but to stress tolerance. The genetic tuning of glycolytic flux may reflect an adaptive trade-off in a species lacking pyruvate kinase isoforms.


Assuntos
Carbono/metabolismo , Mutação de Sentido Incorreto , Piruvato Quinase/genética , Schizosaccharomyces/crescimento & desenvolvimento , Fermentação , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Glicólise , Estresse Oxidativo , Polimorfismo de Nucleotídeo Único , Proteômica , Piruvato Quinase/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
4.
Methods Mol Biol ; 2049: 263-282, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31602617

RESUMO

All biosynthetically active cells are able to export and import metabolites, the small molecule intermediaries of metabolism. In dense cell populations, this hallmark of cells results in the intercellular exchange of a wide spectrum of metabolites. Such metabolite exchange enables metabolic specialization of individual cells, leading to far reaching biological implications, as a consequence of the intrinsic connection between metabolism and cell physiology. In this chapter, we discuss methods on how to study metabolite exchange interactions by using self-establishing metabolically cooperating communities (SeMeCos) in the budding yeast Saccharomyces cerevisiae. SeMeCos exploit the stochastic segregation of episomes to progressively increase the number of essential metabolic interdependencies in a community that grows out from an initially prototrophic cell. By coupling genotype to metabotype, SeMeCos allow for the tracking of cells while they specialize metabolically and hence the opportunity to study their progressive change in physiology.


Assuntos
Células Eucarióticas/metabolismo , Saccharomyces cerevisiae/metabolismo , Genótipo , Interações Microbianas
5.
Nature ; 572(7768): 249-253, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31367038

RESUMO

Both single and multicellular organisms depend on anti-stress mechanisms that enable them to deal with sudden changes in the environment, including exposure to heat and oxidants. Central to the stress response are dynamic changes in metabolism, such as the transition from the glycolysis to the pentose phosphate pathway-a conserved first-line response to oxidative insults1,2. Here we report a second metabolic adaptation that protects microbial cells in stress situations. The role of the yeast polyamine transporter Tpo1p3-5 in maintaining oxidant resistance is unknown6. However, a proteomic time-course experiment suggests a link to lysine metabolism. We reveal a connection between polyamine and lysine metabolism during stress situations, in the form of a promiscuous enzymatic reaction in which the first enzyme of the polyamine pathway, Spe1p, decarboxylates lysine and forms an alternative polyamine, cadaverine. The reaction proceeds in the presence of extracellular lysine, which is taken up by cells to reach concentrations up to one hundred times higher than those required for growth. Such extensive harvest is not observed for the other amino acids, is dependent on the polyamine pathway and triggers a reprogramming of redox metabolism. As a result, NADPH-which would otherwise be required for lysine biosynthesis-is channelled into glutathione metabolism, leading to a large increase in glutathione concentrations, lower levels of reactive oxygen species and increased oxidant tolerance. Our results show that nutrient uptake occurs not only to enable cell growth, but when the nutrient availability is favourable it also enables cells to reconfigure their metabolism to preventatively mount stress protection.


Assuntos
Antioxidantes/metabolismo , Lisina/metabolismo , Poliaminas/metabolismo , Saccharomyces cerevisiae/metabolismo , Antiporters/metabolismo , Cadaverina/metabolismo , Glutamina/metabolismo , Glutationa/metabolismo , NADP/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Ornitina Descarboxilase/metabolismo , Oxidantes/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
EMBO J ; 38(5)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30737259

RESUMO

Ageing is the biggest risk factor for cardiovascular disease. Cellular senescence, a process driven in part by telomere shortening, has been implicated in age-related tissue dysfunction. Here, we address the question of how senescence is induced in rarely dividing/post-mitotic cardiomyocytes and investigate whether clearance of senescent cells attenuates age-related cardiac dysfunction. During ageing, human and murine cardiomyocytes acquire a senescent-like phenotype characterised by persistent DNA damage at telomere regions that can be driven by mitochondrial dysfunction and crucially can occur independently of cell division and telomere length. Length-independent telomere damage in cardiomyocytes activates the classical senescence-inducing pathways, p21CIP and p16INK4a, and results in a non-canonical senescence-associated secretory phenotype, which is pro-fibrotic and pro-hypertrophic. Pharmacological or genetic clearance of senescent cells in mice alleviates detrimental features of cardiac ageing, including myocardial hypertrophy and fibrosis. Our data describe a mechanism by which senescence can occur and contribute to age-related myocardial dysfunction and in the wider setting to ageing in post-mitotic tissues.


Assuntos
Cardiomegalia/patologia , Senescência Celular , Dano ao DNA , Fibrose/patologia , Mitose , Miócitos Cardíacos/patologia , Encurtamento do Telômero , Envelhecimento , Animais , Cardiomegalia/etiologia , Feminino , Fibrose/etiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Monoaminoxidase/fisiologia , Miócitos Cardíacos/metabolismo , Fenótipo , RNA/fisiologia , Ratos Sprague-Dawley , Telomerase/fisiologia
7.
Aging Cell ; 18(1): e12882, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30468013

RESUMO

Increased activation of the major pro-inflammatory NF-κB pathway leads to numerous age-related diseases, including chronic liver disease (CLD). Rapamycin, an inhibitor of mTOR, extends lifespan and healthspan, potentially via suppression of inflammaging, a process which is partially dependent on NF-κB signalling. However, it is unknown if rapamycin has beneficial effects in the context of compromised NF-κB signalling, such as that which occurs in several age-related chronic diseases. In this study, we investigated whether rapamycin could ameliorate age-associated phenotypes in a mouse model of genetically enhanced NF-κB activity (nfκb1-/- ) characterized by low-grade chronic inflammation, accelerated aging and CLD. We found that, despite showing no beneficial effects in lifespan and inflammaging, rapamycin reduced frailty and improved long-term memory, neuromuscular coordination and tissue architecture. Importantly, markers of cellular senescence, a known driver of age-related pathology, were alleviated in rapamycin-fed animals. Our results indicate that, in conditions of genetically enhanced NF-κB, rapamycin delays aging phenotypes and improves healthspan uncoupled from its role as a suppressor of inflammation.


Assuntos
Inflamação/patologia , Longevidade/fisiologia , NF-kappa B/deficiência , Sirolimo/farmacologia , Animais , Biomarcadores/metabolismo , Longevidade/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo
8.
Nat Protoc ; 12(1): 183-194, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28005069

RESUMO

Mitochondria are not only the 'powerhouse' of the cell; they are also involved in a multitude of processes that include calcium storage, the cell cycle and cell death. Traditional means of investigating mitochondrial importance in a given cellular process have centered upon depletion of mtDNA through chemical or genetic means. Although these methods severely disrupt the mitochondrial electron transport chain, mtDNA-depleted cells still maintain mitochondria and many mitochondrial functions. Here we describe a straightforward protocol to generate mammalian cell populations with low to nondetectable levels of mitochondria. Ectopic expression of the ubiquitin E3 ligase Parkin, combined with short-term mitochondrial uncoupler treatment, stimulates widespread mitophagy and effectively eliminates mitochondria. In this protocol, we explain how to generate Parkin-expressing, mitochondria-depleted cells from scratch in 23 d, as well as offer a variety of methods for confirming mitochondrial clearance. Furthermore, we describe culture conditions to maintain mitochondrial-depleted cells for up to 30 d with minimal loss of viability, for longitudinal studies. This method should prove useful for investigating the importance of mitochondria in a variety of biological processes.


Assuntos
Autofagia , Técnicas de Cultura de Células/métodos , Mitocôndrias , Animais , Linhagem Celular , Senescência Celular , Humanos , Camundongos , Mitocôndrias/metabolismo , Ubiquitina-Proteína Ligases/genética
9.
Mol Cell Oncol ; 3(4): e1162896, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27652315

RESUMO

In a study published in The EMBO Journal, we demonstrated that mitochondria are necessary for the proinflammatory phenotype of senescence. Furthermore, we identified a new senescence-regulatory pathway involving mTOR-dependent mitochondrial biogenesis. These data highlight mitochondria as targets for interventions that counteract the pro-aging effects of senescence while preserving tumor suppression.

11.
Autophagy ; 12(10): 1917-1930, 2016 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-27391408

RESUMO

SQSTM1/p62 (sequestosome 1) selectively targets polyubiquitinated proteins for degradation via macroautophagy and the proteasome. Additionally, SQSTM1 shuttles between the cytoplasmic and nuclear compartments, although its role in the nucleus is relatively unknown. Here, we report that SQSTM1 dynamically associates with DNA damage foci (DDF) and regulates DNA repair. Upon induction of DNA damage SQSTM1 interacts with FLNA (filamin A), which has previously been shown to recruit DNA repair protein RAD51 (RAD51 recombinase) to double-strand breaks and facilitate homologous recombination (HR). SQSTM1 promotes proteasomal degradation of FLNA and RAD51 within the nucleus, resulting in reduced levels of nuclear RAD51 and slower DNA repair. SQSTM1 regulates the ratio between HR and nonhomologous end joining (NHEJ) by promoting the latter at the expense of the former. This SQSTM1-dependent mechanism mediates the effect of macroautophagy on DNA repair. Moreover, nuclear localization of SQSTM1 and its association with DDF increase with aging and are prevented by life-span-extending dietary restriction, suggesting that an imbalance in the mechanism identified here may contribute to aging and age-related diseases.


Assuntos
Autofagia , Reparo do DNA , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Sequestossoma-1/metabolismo , Ubiquitina/metabolismo , Animais , Núcleo Celular/metabolismo , Dano ao DNA , Filaminas , Cinética , Camundongos Endogâmicos C57BL , Modelos Biológicos , Transporte Proteico , Proteólise , Rad51 Recombinase/metabolismo
12.
EMBO J ; 35(7): 724-42, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26848154

RESUMO

Cell senescence is an important tumour suppressor mechanism and driver of ageing. Both functions are dependent on the development of the senescent phenotype, which involves an overproduction of pro-inflammatory and pro-oxidant signals. However, the exact mechanisms regulating these phenotypes remain poorly understood. Here, we show the critical role of mitochondria in cellular senescence. In multiple models of senescence, absence of mitochondria reduced a spectrum of senescence effectors and phenotypes while preserving ATP production via enhanced glycolysis. Global transcriptomic analysis by RNA sequencing revealed that a vast number of senescent-associated changes are dependent on mitochondria, particularly the pro-inflammatory phenotype. Mechanistically, we show that the ATM, Akt and mTORC1 phosphorylation cascade integrates signals from the DNA damage response (DDR) towards PGC-1ß-dependent mitochondrial biogenesis, contributing to aROS-mediated activation of the DDR and cell cycle arrest. Finally, we demonstrate that the reduction in mitochondrial content in vivo, by either mTORC1 inhibition or PGC-1ß deletion, prevents senescence in the ageing mouse liver. Our results suggest that mitochondria are a candidate target for interventions to reduce the deleterious impact of senescence in ageing tissues.


Assuntos
Envelhecimento/fisiologia , Mitocôndrias/fisiologia , Animais , Linhagem Celular , Humanos , Camundongos , Modelos Biológicos , Fenótipo
13.
Am J Physiol Lung Cell Mol Physiol ; 309(10): L1124-37, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26386121

RESUMO

Cellular senescence has been associated with the structural and functional decline observed during physiological lung aging and in chronic obstructive pulmonary disease (COPD). Airway epithelial cells are the first line of defense in the lungs and are important to COPD pathogenesis. However, the mechanisms underlying airway epithelial cell senescence, and particularly the role of telomere dysfunction in this process, are poorly understood. We aimed to investigate telomere dysfunction in airway epithelial cells from patients with COPD, in the aging murine lung and following cigarette smoke exposure. We evaluated colocalization of γ-histone protein 2A.X and telomeres and telomere length in small airway epithelial cells from patients with COPD, during murine lung aging, and following cigarette smoke exposure in vivo and in vitro. We found that telomere-associated DNA damage foci increase in small airway epithelial cells from patients with COPD, without significant telomere shortening detected. With age, telomere-associated foci increase in small airway epithelial cells of the murine lung, which is accelerated by cigarette smoke exposure. Moreover, telomere-associated foci predict age-dependent emphysema, and late-generation Terc null mice, which harbor dysfunctional telomeres, show early-onset emphysema. We found that cigarette smoke accelerates telomere dysfunction via reactive oxygen species in vitro and may be associated with ataxia telangiectasia mutated-dependent secretion of inflammatory cytokines interleukin-6 and -8. We propose that telomeres are highly sensitive to cigarette smoke-induced damage, and telomere dysfunction may underlie decline of lung function observed during aging and in COPD.


Assuntos
Dano ao DNA , Pulmão/patologia , Doença Pulmonar Obstrutiva Crônica/genética , Telômero/genética , Idoso , Envelhecimento , Animais , Estudos de Casos e Controles , Linhagem Celular , Reparo do DNA , Células Epiteliais/patologia , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mucosa Respiratória/patologia , Fumar/efeitos adversos
14.
Biochim Biophys Acta ; 1847(11): 1373-9, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26028303

RESUMO

Cellular senescence entails an irreversible cell-cycle arrest characterised by drastic cytomorphological and metabolic changes. In recent years, the implications of cellular senescence in physiological and pathological settings, such as ageing and cancer, have gained firm ground. It is, therefore, important to understand the mechanisms underpinning the establishment and maintenance of senescence. Age-dependent alterations in cellular metabolic processes are greatly driven by changes in mitochondrial function and homeostasis. Classically, mitochondrial dysfunction has been implicated in cellular senescence mainly by promoting oxidative damage-induced cell-cycle arrest; however, emerging data suggests that other mitochondrial-dependent factors play an important role in the induction of senescent phenotypes. Here we review the role of mitochondrial homeostatic mechanisms, mitochondrial metabolites and ROS generation in the signalling pathways leading to the induction and maintenance of cellular senescence and discuss how this may contribute to the ageing process. This article is part of a Special Issue entitled: Mitochondrial Dysfunction in Aging.


Assuntos
Senescência Celular , Mitocôndrias/fisiologia , Animais , Transporte de Elétrons , Homeostase , Humanos , Espécies Reativas de Oxigênio/metabolismo
15.
Nat Commun ; 2: 4172, 2014 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-24960204

RESUMO

Chronic inflammation is associated with normal and pathological ageing. Here we show that chronic, progressive low-grade inflammation induced by knockout of the nfkb1 subunit of the transcription factor NF-κB induces premature ageing in mice. We also show that these mice have reduced regeneration in liver and gut. nfkb1(-/-) fibroblasts exhibit aggravated cell senescence because of an enhanced autocrine and paracrine feedback through NF-κB, COX-2 and ROS, which stabilizes DNA damage. Preferential accumulation of telomere-dysfunctional senescent cells in nfkb1(-/-) tissues is blocked by anti-inflammatory or antioxidant treatment of mice, and this rescues tissue regenerative potential. Frequencies of senescent cells in liver and intestinal crypts quantitatively predict mean and maximum lifespan in both short- and long-lived mice cohorts. These data indicate that systemic chronic inflammation can accelerate ageing via ROS-mediated exacerbation of telomere dysfunction and cell senescence in the absence of any other genetic or environmental factor.


Assuntos
Senilidade Prematura/genética , Fibroblastos/metabolismo , Inflamação/genética , Regeneração Hepática/genética , Subunidade p50 de NF-kappa B/genética , Homeostase do Telômero/genética , Senilidade Prematura/imunologia , Animais , Senescência Celular/genética , Senescência Celular/imunologia , Doença Crônica , Ciclo-Oxigenase 2/metabolismo , Dano ao DNA/genética , Dano ao DNA/imunologia , Retroalimentação Fisiológica , Fibroblastos/imunologia , Inflamação/imunologia , Regeneração Hepática/imunologia , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/imunologia , Subunidade p50 de NF-kappa B/imunologia , Espécies Reativas de Oxigênio/metabolismo , Regeneração/genética , Regeneração/imunologia , Homeostase do Telômero/imunologia
16.
Longev Healthspan ; 3(1): 1, 2014 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-24472138

RESUMO

Senescence, the state of irreversible cell-cycle arrest, plays paradoxical albeit important roles in vivo: it protects organisms against cancer but also contributes to age-related loss of tissue function. The DNA damage response (DDR) has a central role in cellular senescence. Not only does it contribute to the irreversible loss of replicative capacity but also to the production and secretion of reactive oxygen species (ROS), and bioactive peptides collectively known as the senescence-associated secretory phenotype (SASP). Both ROS and the SASP have been shown to impact on senescence in an autocrine as well as paracrine fashion; however, the underlying mechanisms are not well understood. In this review we describe our current understanding of cellular senescence, examine in detail the intricate pathways linking the DDR, ROS and SASP, and evaluate their impact on the stability of the senescent phenotype.

17.
Methods Mol Biol ; 965: 409-19, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23296674

RESUMO

Cellular senescence, the irreversible loss of replicative capacity, is both a tumor suppressor mechanism and a contributor to the age-related loss of tissue function. However, the role of cellular senescence in vivo has been unclear, mostly because of the absence of cellular markers specific enough to identify the state (senescent or proliferating) of individual cells in tissues. Recently, we have tested the robustness of multiple senescence candidate markers by comparing them to a dynamic stimulation model, which estimates the fraction of senescent cells with high precision. We found that the absence of the proliferation markers Ki67 and PCNA combined with high density DNA damage foci (>5 γH2AX foci per nucleus) was the best quantitative indicator of cellular senescence. In this chapter, we describe protocols for the dual immunofluorescence-based quantification of Ki67/PCNA and γH2AX in both fixed cells and paraffin-embedded tissues.


Assuntos
Senescência Celular , Imunofluorescência/métodos , Animais , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Histonas/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Camundongos , Inclusão em Parafina , Antígeno Nuclear de Célula em Proliferação/metabolismo , Coloração e Rotulagem , Fixação de Tecidos
18.
Nat Commun ; 3: 708, 2012 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-22426229

RESUMO

Telomeres are specialized nucleoprotein structures, which protect chromosome ends and have been implicated in the ageing process. Telomere shortening has been shown to contribute to a persistent DNA damage response (DDR) during replicative senescence, the irreversible loss of division potential of somatic cells. Similarly, persistent DDR foci can be found in stress-induced senescence, although their nature is not understood. Here we show, using immuno-fluorescent in situ hybridization and ChIP, that up to half of the DNA damage foci in stress-induced senescence are located at telomeres irrespective of telomerase activity. Moreover, live-cell imaging experiments reveal that all persistent foci are associated with telomeres. Finally, we report an age-dependent increase in frequencies of telomere-associated foci in gut and liver of mice, occurring irrespectively of telomere length. We conclude that telomeres are important targets for stress in vitro and in vivo and this has important consequences for the ageing process.


Assuntos
Envelhecimento/fisiologia , Dano ao DNA , Estresse Oxidativo , Encurtamento do Telômero/fisiologia , Envelhecimento/genética , Animais , Divisão Celular , Linhagem Celular , Imunoprecipitação da Cromatina , Reparo do DNA , Replicação do DNA , Trato Gastrointestinal/citologia , Humanos , Hibridização in Situ Fluorescente , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/metabolismo , Encurtamento do Telômero/genética
19.
EMBO J ; 29(20): 3531-43, 2010 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-20852589

RESUMO

Accurate chromosome segregation during mitosis requires precise coordination of various processes, such as chromosome alignment, maturation of proper kinetochore-microtubule (kMT) attachments, correction of erroneous attachments, and silencing of the spindle assembly checkpoint (SAC). How these fundamental aspects of mitosis are coordinately and temporally regulated is poorly understood. In this study, we show that the temporal regulation of kMT attachments by CLASP1, astrin and Kif2b is central to mitotic progression and chromosome segregation fidelity. In early mitosis, a Kif2b-CLASP1 complex is recruited to kinetochores to promote chromosome movement, kMT turnover, correction of attachment errors, and maintenance of SAC signalling. However, during metaphase, this complex is replaced by an astrin-CLASP1 complex, which promotes kMT stability, chromosome alignment, and silencing of the SAC. We show that these two complexes are differentially recruited to kinetochores and are mutually exclusive. We also show that other kinetochore proteins, such as Kif18a, affect kMT attachments and chromosome movement through these proteins. Thus, CLASP1-astrin-Kif2b complex act as a central switch at kinetochores that defines mitotic progression and promotes fidelity by temporally regulating kMT attachments.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinesina/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose/fisiologia , Fuso Acromático/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proteína B de Centrômero/genética , Proteína B de Centrômero/metabolismo , Cromossomos/metabolismo , Humanos , Cinesina/genética , Proteínas Associadas aos Microtúbulos/genética , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
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