RESUMO
Cells infected with poliovirus exhibit a rapid inhibition of protein secretion and disruption of the Golgi complex. Neither the precise step at which the virus inhibits protein secretion nor the fate of the Golgi complex during infection has been determined. We find that transport-vesicle exit from the endoplasmic reticulum (ER) and trafficking to the ER-Golgi intermediate compartment (ERGIC) are unaffected in the poliovirus-infected cell. By contrast, poliovirus infection blocks transport from the ERGIC to the Golgi complex. Poliovirus infection also induces fragmentation of the Golgi complex resulting in diffuse distribution of both large and small vesicles throughout the cell. Pre-treatment with nocodazole prevents complete fragmentation, indicating that microtubules are required for poliovirus-induced Golgi dispersion. However, virally induced inhibition of the secretory pathway is not affected by nocodazole, and Golgi dispersion was found to occur during infection with mutant viruses with reduce ability to inhibit protein secretion. We conclude that the dispersion of the Golgi complex is not in itself the cause of inhibition of traffic between the ERGIC and the Golgi. Instead, these phenomena are independent effects of poliovirus infection on the host secretory complex.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Poliomielite/metabolismo , Poliovirus/metabolismo , Animais , Antineoplásicos/farmacologia , Células COS , Chlorocebus aethiops , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Nocodazol/farmacologia , Poliomielite/patologia , Poliovirus/ultraestrutura , Transporte Proteico/efeitos dos fármacos , Proteínas/metabolismoRESUMO
The CellCard system enables the analysis of multiple cell types within a single microtiter well. In doing so, the CellCard system not only determines the effect of an experimental condition on a cell type of interest, but also the relative selectivity of that response across nine other cell types. In addition, this approach of cellular multiplexing is a means of miniaturization without the necessity of microfluidic devices. The standard 96-well plate generates ten 96-well plates of data (or, the equivalent of a 960-well plate). Taken together, the CellCard technology enables multiple cell types to be assayed within a single microtiter well allowing for the simultaneous determination of cellular activity and compound selectivity. This chapter will describe a method by which multiple cell types can be simultaneously assayed for biological parameters of interest.
Assuntos
Análise Serial de Tecidos/métodos , Animais , Células Cultivadas , Humanos , Processamento de Imagem Assistida por Computador , Coloração e Rotulagem , Análise Serial de Tecidos/instrumentaçãoRESUMO
High-content screening technologies utilize assays that monitor and quantify multiple cellular events. These assays are typically performed on a single cell type with automated microscopy and image analysis. However, in order to better understand the selectivity of a compound across multiple cell lines, these types of assay must be run serially, which is time consuming. The CellCard System developed by Vitra Bioscience enables multiple cell types to be assayed within a single microtiter well, thereby enabling the simultaneous determination of cellular responses across ten cell types. This multiplexed approach could address the demand for assay capacity, increase the quality of the biologic data, reduce timelines, and improve cost-effectiveness in hit identification and lead evaluation. The authors have carried out an in-depth evaluation of this technology platform using ten cancer cell lines and a library of compounds that affect cellular growth through different mechanisms. Multiple assays were used to investigate the compound effects on membrane integrity, cell cycle progression and apoptosis. In this technology review, the authors discuss personal experience with assay validation, data analysis, results such as cell type-specific compound effects, and the potential application of the CellCard System in drug discovery.
Assuntos
Bioensaio/métodos , Fenômenos Fisiológicos Celulares , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , HumanosRESUMO
Advances in high throughput screening technologies have led to the identification of many small molecules, "hits", with activities toward the target of interest. And, as the screening technologies become faster and more robust, the rate at which the molecules are identified continues to increase. This evolution of high throughput screening technologies has generated a significant strain on the laboratories involved with the downstream profiling of these hits using cell-based assays. The CellCard System, by enabling multiple targets and/or cell lines to be assayed simultaneously within a single well, provides a platform on which selectivity screening can be quickly and robustly performed. Here we describe two case studies using the beta-lactamase and beta-galactosidase reporter gene systems to characterize G protein-coupled receptor agonist activity. Using these examples we demonstrate how the implementation of this technology enables assay miniaturization without micro-fluidic devices as well as how the inclusion of intra-well controls can provide a means of data quality assessment within each well.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Animais , Bioensaio , Células CHO , Linhagem Celular , Cricetinae , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/instrumentação , Genes Reporter , Humanos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismo , beta-Lactamases/efeitos dos fármacos , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
The authors have developed a cellular analysis platform, based on encoded microcarriers, that enables the multiplexed analysis of a diverse range of cellular assays. At the core of this technology are classes of microcarriers that have unique, identifiable codes that are deciphered using CCD-based imaging and subsequent image analysis. The platform is compatible with a wide variety of cellular imaging-based assays, including calcium flux, reporter gene activation, cytotoxicity, and proliferation. In addition, the platform is compatible with both colorimetric and fluorescent readouts. Notably, this technology has the unique ability to multiplex different cell lines in a single microplate well, enabling scientists to perform assays and data analysis in novel ways.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Biologia Molecular/métodos , Animais , Células CHO , Cálcio/metabolismo , Carbacol/farmacologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Células Imobilizadas , Cricetinae , Humanos , Processamento de Imagem Assistida por Computador , Calidina/farmacologia , Microscopia/instrumentação , Microscopia/métodos , Tamanho da Partícula , Receptor B2 da Bradicinina/análise , Receptor B2 da Bradicinina/efeitos dos fármacos , Receptor B2 da Bradicinina/genética , Receptores Acoplados a Proteínas G/análise , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores Acoplados a Proteínas G/genética , Receptores Muscarínicos/análise , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/genética , Transdução de Sinais , Testes de ToxicidadeRESUMO
Viral replication depends on specific interactions with host factors. For example, poliovirus RNA replication requires association with intracellular membranes. Brefeldin A (BFA), which induces a major rearrangement of the cellular secretory apparatus, is a potent inhibitor of poliovirus RNA replication. Most aspects governing the relationship between viral replication complex and the host membranes remain poorly defined. To explore these interactions, we used a genetic approach and isolated BFA-resistant poliovirus variants. Mutations within viral proteins 2C and 3A render poliovirus resistant to BFA. In the absence of BFA, viruses containing either or both of these mutations replicated similarly to wild type. In the presence of BFA, viruses carrying a single mutation in 2C or 3A exhibited an intermediate-growth phenotype, while the double mutant was fully resistant. The viral proteins 2C and 3A have critical roles in both RNA replication and vesicle formation. The identification of BFA resistant mutants may facilitate the identification of cellular membrane-associated proteins necessary for induction of vesicle formation and RNA replication. Importantly, our data underscore the dramatic plasticity of the host-virus interactions required for successful viral replication.
Assuntos
Antivirais/farmacologia , Brefeldina A/farmacologia , Farmacorresistência Viral , Poliovirus/efeitos dos fármacos , Poliovirus/fisiologia , Replicação Viral/efeitos dos fármacos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Vesículas Citoplasmáticas/efeitos dos fármacos , Farmacorresistência Viral/genética , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Células HeLa , Interações Hospedeiro-Parasita , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Cinética , Fenótipo , Poliovirus/genética , Replicon/efeitos dos fármacos , Replicon/genética , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
The desire for more biologically relevant data from primary screening has resulted in a dramatic increase of cell-based assays in HTS labs. Consequently, new cell-array technologies are being developed to increase the quality and quantity of data emerging from such screens. These technologies take the form of both positional and non-positional formats, each with their own advantages. Notably, screens using these technologies generate databases of high-quality data that can be analyzed in ways currently not possible. The power of cell-based assays combined with new array and analytical technologies will enable the condensation of serial drug discovery processes, thereby decreasing the time and cost of taking a hit compound into clinical trials. Here, we compare array strategies being developed towards the goal of integrating multiplexed cell-based assays into HTS.