RESUMO
Cells have evolved biomolecular networks that process and respond to changing chemical environments. Understanding how complex protein interactions give rise to emergent network properties requires time-resolved analysis of cellular response under a large number of genetic perturbations and chemical environments. To date, the lack of technologies for scalable cell analysis under well-controlled and time-varying conditions has made such global studies either impossible or impractical. To address this need, we have developed a high-throughput microfluidic imaging platform for single-cell studies of network response under hundreds of combined genetic perturbations and time-varying stimulant sequences. Our platform combines programmable on-chip mixing and perfusion with high-throughput image acquisition and processing to perform 256 simultaneous time-lapse live-cell imaging experiments. Nonadherent cells are captured in an array of 2,048 microfluidic cell traps to allow for the imaging of eight different genotypes over 12 h and in response to 32 unique sequences of stimulation, generating a total of 49,000 images per run. Using 12 devices, we carried out >3,000 live-cell imaging experiments to investigate the mating pheromone response in Saccharomyces cerevisiae under combined genetic perturbations and changing environmental conditions. Comprehensive analysis of 11 deletion mutants reveals both distinct thresholds for morphological switching and new dynamic phenotypes that are not observed in static conditions. For example, kss1Delta, fus3Delta, msg5Delta, and ptp2Delta mutants exhibit distinctive stimulus-frequency-dependent signaling phenotypes, implicating their role in filtering and network memory. The combination of parallel microfluidic control with high-throughput imaging provides a powerful tool for systems-level studies of single-cell decision making.
Assuntos
Imageamento Tridimensional/instrumentação , Sistema de Sinalização das MAP Quinases , Microfluídica/instrumentação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator de Acasalamento , Mutação/genética , Peptídeos/farmacologia , Fenótipo , Feromônios/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologiaRESUMO
Microarray-based gene expression analysis identified genes showing ploidy-dependent expression in isogenic Saccharomyces cerevisiae strains that varied in ploidy from haploid to tetraploid. These genes were induced or repressed in proportion to the number of chromosome sets, regardless of the mating type. Ploidy-dependent repression of some G1 cyclins can explain the greater cell size associated with higher ploidies, and suggests ploidy-dependent modifications of cell cycle progression. Moreover, ploidy regulation of the FLO11 gene had direct consequences for yeast development.
Assuntos
Regulação Fúngica da Expressão Gênica , Ploidias , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Quitinases/genética , Ciclinas/genética , Proteínas Fúngicas/genética , Fase G1 , Haploidia , Lipoproteínas/genética , Lipoproteínas/fisiologia , Fator de Acasalamento , Glicoproteínas de Membrana , Proteínas de Membrana/genética , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/genética , Peptídeos/fisiologia , Feromônios , Poliploidia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/genéticaRESUMO
Previous studies in which chicken and human c-Src were overexpressed in chicken and rodent cells have indicated that overexpression of wild-type c-Src can not induce complete neoplastic transformation. However, studies with v-Src mutants have demonstrated that species-specific differences can play a significant role in transforming activity. Here we show that, in contrast to chicken c-Src, overexpressed mouse c-Src can induce significant anchorage-independent growth and tumorigenicity when transfected into NIH3T3 mouse cells. The biochemical cause for this difference is unknown. In particular, the protein-tyrosine kinase activities of chicken and mouse c-Src appear to be similar. This result is consistent with the hypothesis that v-Src-induced transformation results from perturbation of signalling pathways modulated by c-Src and highlights the need for caution in controlling for potential species-specific differences in studies of c-Src function.
Assuntos
Transformação Celular Neoplásica/genética , Proteínas Proto-Oncogênicas pp60(c-src)/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Camundongos , Dados de Sequência Molecular , Especificidade da Espécie , TransfecçãoRESUMO
The heat shock response and the heat shock proteins have been conserved across evolution. In Escherichia coli, the heat shock response is positively regulated by the sigma 32 transcriptional factor and negatively regulated by a subset of the heat shock proteins themselves. In an effort to understand the regulation of the heat shock response, we have purified the sigma 32 polypeptide to homogeneity. During the purification procedure, we found that a large fraction of the overexpressed sigma 32 polypeptide copurified with the universally conserved DnaK heat shock protein (the prokaryotic equivalent of the 70-kDa heat shock protein, HSP70). Further experiments established that purified sigma 32 bound to DnaK and that this complex was disrupted in the presence of ATP. Consistent with the fact that dnaK756 mutant bacteria overexpress heat shock proteins at all temperatures, purified DnaK756 mutant protein did not appreciably bind to sigma 32.