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1.
Elife ; 42015 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-25626168

RESUMO

Motor proteins of the conserved kinesin-14 family have important roles in mitotic spindle organization and chromosome segregation. Previous studies have indicated that kinesin-14 motors are non-processive enzymes, working in the context of multi-motor ensembles that collectively organize microtubule networks. In this study, we show that the yeast kinesin-14 Kar3 generates processive movement as a heterodimer with the non-motor proteins Cik1 or Vik1. By analyzing the single-molecule properties of engineered motors, we demonstrate that the non-catalytic domain has a key role in the motility mechanism by acting as a 'foothold' that allows Kar3 to bias translocation towards the minus end. This mechanism rivals the speed and run length of conventional motors, can support transport of the Ndc80 complex in vitro and is critical for Kar3 function in vivo. Our findings provide an example for a non-conventional translocation mechanism and can explain how Kar3 substitutes for key functions of Dynein in the yeast nucleus.


Assuntos
Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Domínio Catalítico , Dimerização , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Frações Subcelulares/metabolismo
2.
Nat Genet ; 46(9): 973-981, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25108384

RESUMO

Polycomb/Trithorax response elements (PRE/TREs) can switch their function reversibly between silencing and activation by mechanisms that are poorly understood. Here we show that a switch in forward and reverse noncoding transcription from the Drosophila melanogaster vestigial (vg) PRE/TRE switches the status of the element between silencing (induced by the forward strand) and activation (induced by the reverse strand). In vitro, both noncoding RNAs inhibit PRC2 histone methyltransferase activity, but, in vivo, only the reverse strand binds PRC2. Overexpression of the reverse strand evicts PRC2 from chromatin and inhibits its enzymatic activity. We propose that the interaction of RNAs with PRC2 is differentially regulated in vivo, allowing regulated inhibition of local PRC2 activity. Genome-wide analysis shows that strand switching of noncoding RNAs occurs at several hundred Polycomb-binding sites in fly and vertebrate genomes. This work identifies a previously unreported and potentially widespread class of PRE/TREs that switch function by switching the direction of noncoding RNA transcription.


Assuntos
Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/genética , Genes de Troca , Proteínas do Grupo Polycomb/genética , RNA não Traduzido , Elementos de Resposta , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Cromatina/genética , Proteínas de Ligação a DNA/genética , Drosophila melanogaster , Genoma de Inseto , Histona-Lisina N-Metiltransferase/genética , Dados de Sequência Molecular , Fatores de Transcrição/genética
3.
Nat Commun ; 5: 3056, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24445999

RESUMO

Autophagy is a mechanism by which starving cells can control their energy requirements and metabolic states, thus facilitating the survival of cells in stressful environments, in particular in the pathogenesis of cancer. Here we report that tissue-specific inactivation of Atg5, essential for the formation of autophagosomes, markedly impairs the progression of KRas(G12D)-driven lung cancer, resulting in a significant survival advantage of tumour-bearing mice. Autophagy-defective lung cancers exhibit impaired mitochondrial energy homoeostasis, oxidative stress and a constitutively active DNA damage response. Genetic deletion of the tumour suppressor p53 reinstates cancer progression of autophagy-deficient tumours. Although there is improved survival, the onset of Atg5-mutant KRas(G12D)-driven lung tumours is markedly accelerated. Mechanistically, increased oncogenesis maps to regulatory T cells. These results demonstrate that, in KRas(G12D)-driven lung cancer, Atg5-regulated autophagy accelerates tumour progression; however, autophagy also represses early oncogenesis, suggesting a link between deregulated autophagy and regulatory T cell controlled anticancer immunity.


Assuntos
Autofagia/fisiologia , Modelos Animais de Doenças , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/fisiopatologia , Proteínas Associadas aos Microtúbulos/fisiologia , Animais , Proteína 5 Relacionada à Autofagia , Progressão da Doença , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas aos Microtúbulos/genética , Mutação/genética , Linfócitos T Reguladores/patologia , Linfócitos T Reguladores/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologia
4.
Nat Protoc ; 8(6): 1088-1099, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23660757

RESUMO

Elegant tools are available for the genetic analysis of neural stem cell lineages in Drosophila, but a methodology for purifying stem cells and their differentiated progeny for transcriptome analysis is currently missing. Previous attempts to overcome this problem either involved using RNA isolated from whole larval brain tissue or co-transcriptional in vivo mRNA tagging. As both methods have limited cell type specificity, we developed a protocol for the isolation of Drosophila neural stem cells (neuroblasts, NBs) and their differentiated sibling cells by FACS. We dissected larval brains from fly strains expressing GFP under the control of a NB lineage-specific GAL4 line. Upon dissociation, we made use of differences in GFP intensity and cell size to separate NBs and neurons. The resulting cell populations are over 98% pure and can readily be used for live imaging or gene expression analysis. Our method is optimized for neural stem cells, but it can also be applied to other Drosophila cell types. Primary cell suspensions and sorted cell populations can be obtained within 1 d; material for deep-sequencing library preparation can be obtained within 4 d.


Assuntos
Drosophila/citologia , Citometria de Fluxo/métodos , Células-Tronco Neurais/citologia , Animais , Técnicas de Cultura de Células/métodos , Proteínas de Fluorescência Verde/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Larva/citologia
5.
Cell Stem Cell ; 9(6): 563-74, 2011 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-22136931

RESUMO

All somatic mammalian cells carry two copies of chromosomes (diploidy), whereas organisms with a single copy of their genome, such as yeast, provide a basis for recessive genetics. Here we report the generation of haploid mouse ESC lines from parthenogenetic embryos. These cells carry 20 chromosomes, express stem cell markers, and develop into all germ layers in vitro and in vivo. We also developed a reversible mutagenesis protocol that allows saturated genetic recessive screens and results in homozygous alleles. This system allowed us to generate a knockout cell line for the microRNA processing enzyme Drosha. In a forward genetic screen, we identified Gpr107 as a molecule essential for killing by ricin, a toxin being used as a bioweapon. Our results open the possibility of combining the power of a haploid genome with pluripotency of embryonic stem cells to uncover fundamental biological processes in defined cell types at a genomic scale.


Assuntos
Células-Tronco Embrionárias/fisiologia , Haploidia , Genética Reversa/métodos , Animais , Biomarcadores/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Estudo de Associação Genômica Ampla , Camundongos , Camundongos Endogâmicos C57BL , Partenogênese/genética , Ricina/toxicidade
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