RESUMO
RNA interference (RNAi) has become a powerful technique for reverse genetics and drug discovery, and in both of these areas large-scale high-throughput RNAi screens are commonly performed. The statistical techniques used to analyze these screens are frequently borrowed directly from small-molecule screening; however, small-molecule and RNAi data characteristics differ in meaningful ways. We examine the similarities and differences between RNAi and small-molecule screens, highlighting particular characteristics of RNAi screen data that must be addressed during analysis. Additionally, we provide guidance on selection of analysis techniques in the context of a sample workflow.
Assuntos
Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Projetos de Pesquisa/estatística & dados numéricos , Bibliotecas de Moléculas Pequenas , Animais , Simulação por Computador , Modelos EstatísticosRESUMO
Heterotrimeric G proteins act during signal transduction in response to extracellular ligands. They are also required for spindle orientation and cell polarity during asymmetric cell division. We show here that, in Drosophila, both functions require the Galpha interaction partner Ric-8. Drosophila Ric-8 is a cytoplasmic protein that binds both the GDP- and GTP-bound form of the G-protein alpha-subunit Galphai. In ric-8 mutants, neither Galphai nor its associated beta-subunit Gbeta13F are localized at the plasma membrane, which leads to their degradation in the cytosol. During asymmetric cell division, this leads to various defects: apico-basal polarity is not maintained, mitotic spindles are misoriented and the size of the two daughter cells becomes nearly equal. ric-8 mutants also have defects in gastrulation that resemble mutants in the Galpha protein concertina or the extracellular ligand foldedgastrulation. Our results indicate a model in which both receptor-dependent and receptor-independent G-protein functions are executed at the plasma membrane and require the Ric-8 protein.
Assuntos
Divisão Celular/fisiologia , Membrana Celular/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Animais , Animais Geneticamente Modificados , Polaridade Celular , Drosophila/embriologia , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Proteínas Heterotriméricas de Ligação ao GTP/genética , Neurônios/metabolismo , Neurônios/fisiologia , Neurônios/ultraestruturaRESUMO
During mammalian neurogenesis, progenitor cells can divide with the mitotic spindle oriented parallel or perpendicular to the surface of the neuroepithelium. Perpendicular divisions are more likely to be asymmetric and generate one progenitor and one neuronal precursor. Whether the orientation of the mitotic spindle actually determines their asymmetric outcome is unclear. Here, we characterize a mammalian homolog of Inscuteable (mInsc), a key regulator of spindle orientation in Drosophila. mInsc is expressed temporally and spatially in a manner that suggests a role in orienting the mitotic spindle in the developing nervous system. Using retroviral RNAi in rat retinal explants, we show that downregulation of mInsc inhibits vertical divisions. This results in enhanced proliferation, consistent with a higher frequency of symmetric divisions generating two proliferating cells. Our results suggest that the orientation of neural progenitor divisions is important for cell fate specification in the retina and determines their symmetric or asymmetric outcome.
Assuntos
Proteínas do Citoesqueleto/fisiologia , Proteínas de Drosophila/fisiologia , Neuropeptídeos/fisiologia , Retina/embriologia , Retina/crescimento & desenvolvimento , Fuso Acromático/fisiologia , Animais , Animais Recém-Nascidos , Células COS , Diferenciação Celular/fisiologia , Proliferação de Células , Chlorocebus aethiops , Proteínas do Citoesqueleto/genética , Proteínas de Drosophila/genética , Desenvolvimento Embrionário/fisiologia , Evolução Molecular , Camundongos , Células NIH 3T3 , Neurônios/citologia , Neuropeptídeos/genética , Células Fotorreceptoras/citologia , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Retina/citologia , Células-Tronco/citologiaRESUMO
Deubiquitinating enzymes are now emerging as potential therapeutic targets that control many cellular processes, but few have been demonstrated to control cell motility. Here, we show that ubiquitin-specific protease 17 (USP17) is rapidly and transiently induced in response to chemokines SDF-1/CXCL12 and IL-8/CXCL8 in both primary cells and cell lines, and that its depletion completely blocks chemokine-induced cell migration and cytoskeletal rearrangements. Using live cell imaging, we demonstrate that USP17 is required for both elongated and amoeboid motility, in addition to chemotaxis. USP17 has previously been reported to disrupt Ras localization and we now find that USP17 depletion blocks chemokine-induced subcellular relocalization of GTPases Cdc42, Rac and RhoA, which are GTPases essential for cell motility. Collectively, these results demonstrate that USP17 has a critical role in cell migration and may be a useful drug target for both inflammatory and metastatic disease.
Assuntos
Movimento Celular/fisiologia , Endopeptidases/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Membrana Celular/metabolismo , Quimiocina CXCL12/metabolismo , Quimiotaxia/fisiologia , Citoesqueleto/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Ativação Enzimática , Células HeLa , Humanos , Interleucina-8/metabolismo , Transporte Proteico , Proteínas rho de Ligação ao GTP/análiseRESUMO
High-content screening (HCS) technologies are becoming increasingly used in both large-scale drug discovery and basic research programs. These automated imaging and analysis technologies enable the researcher to elucidate the complex biology that underlies the functions of genes, proteins, and other biomolecules at the cellular level. HCS combines the power of automated digital microscopy and advanced software-based image analysis algorithms to detect and quantify biological changes in cells and tissues. This technology is a particularly powerful tool when used to interrogate the cellular effects of exogenously applied agents such as RNAi and/or small molecules. HCS allows for the evaluation of cellular perturbations that occur both at the level of the single cell and within cellular populations. In a multivariate approach, multiple cellular parameters are collected, allowing for more complex analysis. However, in these scenarios, data flow and management still represent substantial bottlenecks in HCS projects. HCS data include a diversity of information from multiple sources such as details pertaining to screening libraries (e.g., siRNA and small molecules), image stacks acquired from automated microscopes (of which there may be up to several million), and the image analysis data. From this, postprocessing algorithms are required to generate statistical, quality control bioinformatic information and ultimately a final hit list. To accomplish these individual tasks, numerous tools can be used to perform each analytical step; however, management of the entire information flow currently requires the use of commercially available proprietary software, the scope of which is often limited, or bespoke customized scripts. In this article, the authors introduce an open-source research tool that allows for the management of the entire data flow of the HCS data chain, by handling and linking information and providing many powerful postprocessing and visualization tools.
Assuntos
Bioensaio/métodos , Ensaios de Triagem em Larga Escala/métodos , Software , Fluxo de Trabalho , Estatística como AssuntoRESUMO
RNA interfering (RNAi) screening strategies offer the potential to elucidate the signaling pathways that regulate integrin and adhesion receptor-mediated changes in T lymphocyte morphology. Of crucial importance, however, is the definition of key sets of parameters that will provide accurate, quantitative, and nonredundant information to flag relevant hits in such assays. In this study, the authors have used an image-based high-content analysis (HCA) technology platform and a panel of 24 pharmacological inhibitors, at a range of concentrations, to define key sets of parameters that enables sensitive and quantitative effects on integrin (LFA-1)-mediated lymphocyte morphology to be evaluated. In particular, multiparametric analysis of lymphocyte morphology that was based on intracellular staining of both the F-actin and alpha-tubulin cytoskeleton resulted in improved ability to discriminate morphological behavior compared to F-actin staining alone. Morphological and fluorescence intensity/distribution profiling of pharmacologically treated lymphocytes stimulated with integrin (LFA-1) and adhesion receptors (CD44) also revealed notable differences in their sensitivity to inhibitors. The assay described here may be used in HCA strategies such as RNAi screening assays to elucidate the signaling pathways and molecules that regulate integrin/adhesion receptor-mediated T lymphocyte polarization.
Assuntos
Polaridade Celular , Transdução de Sinais/fisiologia , Linfócitos T/citologia , Actinas/metabolismo , Animais , Linhagem Celular , Citoesqueleto/metabolismo , Humanos , Antígeno-1 Associado à Função Linfocitária/metabolismo , Linfócitos T/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Activator of G-protein signaling 3 (AGS3) has a modular domain structure consisting of seven tetratricopeptide repeats (TPRs) and four G-protein regulatory (GPR) motifs. Each GPR motif binds to the alpha subunit of Gi/Go (Gialpha > Goalpha) stabilizing the GDP-bound conformation of Galpha and apparently competing with Gbetagamma for GalphaGDP binding. As an initial approach to identify regulatory mechanisms for AGS3-G-protein interactions, a yeast two-hybrid screen was initiated using the TPR and linker region of AGS3 as bait. This screen identified the serine/threonine kinase LKB1, which is involved in the regulation of cell cycle progression and polarity. Protein interaction assays in mammalian systems using transfected cells or brain lysate indicated the regulated formation of a protein complex consisting of LKB1, AGS3, and G-proteins. The interaction between AGS3 and LKB1 was also observed with orthologous proteins in Drosophila where both proteins are involved in cell polarity. LKB1 immunoprecipitates from COS7 cells transfected with LKB1 phosphorylated the GPR domains of AGS3 and the related protein LGN but not the AGS3-TPR domain. GPR domain phosphorylation was completely blocked by a consensus GPR motif peptide, and placement of a phosphate moiety within a consensus GPR motif reduced the ability of the peptide to interact with G-proteins. These data suggest that phosphorylation of GPR domains may be a general mechanism regulating the interaction of GPR-containing proteins with G-proteins. Such a mechanism may be of particular note in regard to localized signal processing in the plasma membrane involving G-protein subunits and/or intracellular functions regulated by heterotrimeric G-proteins that occur independently of a typical G-protein-coupled receptor.