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BACKGROUND: Systems biology and functional genomics require genome-wide datasets and resources. Complete sets of cloned open reading frames (ORFs) have been made for about a dozen bacterial species and allow researchers to express and study complete proteomes in a high-throughput fashion. RESULTS: We have constructed an open reading frame (ORFeome) collection of 3974 or 94% of the known Escherichia coli K-12 ORFs in Gateway entry vector pENTR/Zeo. The collection has been used for protein expression and protein interaction studies. For example, we have compared interactions among YgjD, YjeE and YeaZ proteins in E. coli, Streptococcus pneumoniae, and Staphylococcus aureus. We also compare this ORFeome with other Gateway-compatible bacterial ORFeomes and show its utility for comparative functional genomics. CONCLUSIONS: The E. coli ORFeome provides a useful resource for functional genomics and other areas of protein research in a highly flexible format. Our comparison with other ORFeomes makes comparative analyses straighforward and facilitates direct comparisons of many proteins across many genomes.
Assuntos
Escherichia coli K12/genética , Fases de Leitura Aberta , Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Análise de Sequência de DNA , Staphylococcus aureus/genética , Streptococcus pneumoniae/genéticaRESUMO
Studies of chemotaxis in the social amoeba Dictyostelium discoideum have revealed numerous conserved signaling networks that are activated by chemoattractants. In the presence of a uniformly distributed stimulus, these pathways are transiently activated, but in a gradient they are activated persistently and can be localized to either the front or the back of the cell. Recent studies have begun to elucidate how chemoattractant signaling regulates the three main components of chemotaxis: directional sensing, pseudopod extension, and polarization.
Assuntos
Quimiotaxia , Dictyostelium/fisiologia , Transdução de Sinais , Animais , Polaridade Celular/fisiologia , Pseudópodes/fisiologiaAssuntos
Quimiotaxia , Neutrófilos/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/fisiologia , Animais , Classe Ib de Fosfatidilinositol 3-Quinase , Humanos , Inositol Polifosfato 5-Fosfatases , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Camundongos , Modelos Biológicos , Neutrófilos/enzimologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/fisiologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismoRESUMO
Efforts to map the Escherichia coli interactome have identified several hundred macromolecular complexes, but direct binary protein-protein interactions (PPIs) have not been surveyed on a large scale. Here we performed yeast two-hybrid screens of 3,305 baits against 3,606 preys (â¼70% of the E. coli proteome) in duplicate to generate a map of 2,234 interactions, which approximately doubles the number of known binary PPIs in E. coli. Integration of binary PPI and genetic-interaction data revealed functional dependencies among components involved in cellular processes, including envelope integrity, flagellum assembly and protein quality control. Many of the binary interactions that we could map in multiprotein complexes were informative regarding internal topology of complexes and indicated that interactions in complexes are substantially more conserved than those interactions connecting different complexes. This resource will be useful for inferring bacterial gene function and provides a draft reference of the basic physical wiring network of this evolutionarily important model microbe.
Assuntos
Proteínas de Escherichia coli , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/fisiologia , Proteômica/métodos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Development of effective quantitative indicators and methodologies to assess the outcomes of cross-disciplinary collaborative initiatives has the potential to improve scientific program management and scientific output. This article highlights an example of a prospective evaluation that has been developed to monitor and improve progress of the National Cancer Institute Physical Sciences-Oncology Centers (PS-OC) program. Study data, including collaboration information, was captured through progress reports and compiled using the web-based analytic database: Interdisciplinary Team Reporting, Analysis, and Query Resource. Analysis of collaborations was further supported by data from the Thomson Reuters Web of Science database, MEDLINE database, and a web-based survey. Integration of novel and standard data sources was augmented by the development of automated methods to mine investigator pre-award publications, assign investigator disciplines, and distinguish cross-disciplinary publication content. The results highlight increases in cross-disciplinary authorship collaborations from pre- to post-award years among the primary investigators and confirm that a majority of cross-disciplinary collaborations have resulted in publications with cross-disciplinary content that rank in the top third of their field. With these evaluation data, PS-OC Program officials have provided ongoing feedback to participating investigators to improve center productivity and thereby facilitate a more successful initiative. Future analysis will continue to expand these methods and metrics to adapt to new advances in research evaluation and changes in the program.
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BACKGROUND: The ability of a cell to change shape is crucial for the proper function of many cellular processes, including cell migration. One type of cell migration, referred to as amoeboid motility, involves alternating cycles of morphological expansion and retraction. Traditionally, this process has been characterized by a number of parameters providing global information about shape changes, which are insufficient to distinguish phenotypes based on local pseudopodial activities that typify amoeboid motility. RESULTS: We developed a method that automatically detects and characterizes pseudopodial behavior of cells. The method uses skeletonization, a technique from morphological image processing to reduce a shape into a series of connected lines. It involves a series of automatic algorithms including image segmentation, boundary smoothing, skeletonization and branch pruning, and takes into account the cell shape changes between successive frames to detect protrusion and retraction activities. In addition, the activities are clustered into different groups, each representing the protruding and retracting history of an individual pseudopod. CONCLUSIONS: We illustrate the algorithms on movies of chemotaxing Dictyostelium cells and show that our method makes it possible to capture the spatial and temporal dynamics as well as the stochastic features of the pseudopodial behavior. Thus, the method provides a powerful tool for investigating amoeboid motility.
Assuntos
Osso e Ossos/patologia , Dictyostelium/fisiologia , Algoritmos , Amoeba/fisiologia , Animais , Automação , Movimento Celular , Forma Celular , Análise por Conglomerados , Processamento de Imagem Assistida por Computador , Modelos Biológicos , Fenótipo , Pseudópodes , Especificidade da Espécie , Biologia de SistemasRESUMO
In a forward genetic screen for chemotaxis mutants in Dictyostelium discoideum, we identified a loss-of-function mutation, designated tsunami, encoding a homolog of the Fused kinase. Cells lacking tsuA function could not effectively perform chemotaxis and were unable to become polarized or correctly orient pseudopods in chemotactic gradients. While tsuA(-) cells were able to couple receptor occupancy to phosphatidylinositol (3,4,5) trisphosphate (PIP3) production and actin polymerization, the PIP3 response was prolonged and basal F-actin levels were increased. Interestingly, TsuA localizes to the microtubule network and puncta mainly found at the cell periphery. Analysis of the gene uncovered a novel C-terminal domain that we designated the Tsunami Homology (TH) domain. Both the kinase domain and the TH domain are required to rescue the phenotypic defects of tsuA(-) cells. While kinase activity is not required for localization to microtubules, the TH domain is essential. Thus, localization of kinase activity to microtubules is critical for TsuA function. We propose that functions in association with the microtubule network may underlie the divergent roles of Fused kinase proteins in different organisms.
Assuntos
Movimento Celular , Polaridade Celular , Quimiotaxia/fisiologia , Dictyostelium/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular , AMP Cíclico/metabolismo , Perfilação da Expressão Gênica , Microtúbulos/metabolismo , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Fosfatidilinositóis/metabolismo , Transporte Proteico , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Frações SubcelularesRESUMO
Wnt-signal transduction through beta-catenin is thought to require the inhibition of GSK3 by Frat/GBP. To investigate the role of Frat in mammalian development, we have generated mice with targeted mutations in all three murine Frat homologs. We show that Frat is normally expressed at sites of active Wnt signaling. Surprisingly, Frat-deficient mice do not display gross abnormalities. Moreover, canonical Wnt signaling in primary cells is unaffected by the loss of Frat. These studies show that Frat is not an essential component of the canonical Wnt pathway in higher organisms, despite the strict requirement of Frat/GBP for maternal Wnt signaling in Xenopus.
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Proteínas de Transporte/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Neoplasias/fisiologia , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Western Blotting , Proteínas de Transporte/genética , Citometria de Fluxo , Imunofluorescência , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas , Proteínas WntRESUMO
Cells use an internal compass to sense the direction of chemoattractant gradients. This is used to bias pseudopod extension at the front of the cell and to orient cell polarization. Recent studies have highlighted the important roles played by phosphoinositide-3,4,5-triphosphate and small G proteins, but many questions remain.
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Movimento Celular/fisiologia , Dictyostelium/fisiologia , Pseudópodes/fisiologia , Animais , Fatores QuimiotáticosRESUMO
Previous studies have shown that nuclear levels of glycogen synthase kinase-3 (GSK-3) are dynamically regulated and may affect access of GSK-3 to its substrates. In this study we show that the GSK-3-binding protein Frat/GBP regulates the nuclear export of GSK-3. We show that Frat/GBP contains a nuclear export sequence that promotes its own nuclear export and that of associated GSK-3. Treating cells with leptomycin B increased nuclear levels of endogenous GSK-3 suggesting that an endogenous process targets GSK-3 for nuclear export. To investigate this further, we used two approaches to disrupt the interaction between GSK-3 and endogenous Frat. First we isolated mutants of GSK-3 that selectively interfered with Frat binding and found that these mutants were poorly exported. Second we expressed a peptide that competes with Frat for GSK-3 binding and found that it caused endogenous GSK-3 to accumulate in the nucleus. Together these data suggest that Frat may be the endogenous factor that targets GSK-3 for nuclear export. The dynamic expression patterns of Frat mRNAs together with the role of Frat in mediating GSK-3 nuclear export have important implications for the control of the substrate access of GSK-3 in several signaling pathways.
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Proteínas de Transporte , Regulação Enzimológica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/biossíntese , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas de Neoplasias , Proteínas Proto-Oncogênicas/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Cães , Ácidos Graxos Insaturados/farmacologia , Glutationa Transferase/metabolismo , Camundongos , Microscopia de Fluorescência , Modelos Genéticos , Dados de Sequência Molecular , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Frações Subcelulares/metabolismo , TransfecçãoRESUMO
Glycogen synthase kinase-3 (GSK-3) is a key component of several signaling pathways including those regulated by Wnt and insulin ligands. Specificity in GSK-3 signaling is thought to involve interactions with scaffold proteins that localize GSK-3 regulators and substrates. This report shows that GSK-3 forms a low affinity homodimer that is disrupted by binding to Axin and Frat. Based on the crystal structure of GSK-3, we have used surface-scanning mutagenesis to identify residues that differentially affect GSK-3 interactions. Mutations that disrupt Frat and Axin cluster at the dimer interface explaining their effect on homodimer formation. Loss of the Axin binding site blocks the ability of dominant negative GSK-3 to cause axis duplication in Xenopus embryos. The Axin binding site is conserved within all GSK-3 proteins, and its loss affects both cell motility and gene expression in the nonmetazoan, Dictyostelium. Surprisingly, we find no genetic interaction between a non-Axin-binding GSK-3 mutant and T-cell factor activity, arguing that Axin interactions alone cannot explain the regulation of T-cell factor-mediated gene expression.