RESUMO
Recently, the functionality of under oil open microfluidics was expanded from droplet-based operations to include lateral flow in under oil aqueous channels. However, the resolution of the under oil fluidic channels reported so far is still far from comparable with that of closed-channel microfluidics (millimeters versus micrometers). Here, enabled by exclusive liquid repellency and an under oil sweep technique, open microchannels can now be prepared under oil (rather than in air), which shrinks the channel dimensions up to three orders of magnitude compared to previously reported techniques. Spatial trapping of different cellular samples and advanced control of mass transport (i.e., enhanced upper limit of flow rate, steady flow with passive pumping, and reversible fluidic valves) were achieved with open-channel designs. We apply these functional advances to enable dynamic measurements of dispersion from a pathogenic fungal biofilm. The ensemble of added capabilities reshapes the potential application space for open microfluidics.
RESUMO
Microfluidic platforms are ideal for generating dynamic temporal and spatial perturbations in extracellular environments. Single cells and organisms can be trapped and maintained in microfluidic platforms for long periods of time while their responses to stimuli are measured using appropriate fluorescence reporters and time-lapse microscopy. Such platforms have been used to study problems as diverse as C. elegans olfaction (Chronis et al. Nature Methods 4:727-731, 2007), cancer cell migration (Huang et al. Biomicrofluidics 5:13412, 2011), and E. coli chemotaxis (Ahmed et al. Integr Biol 2:604-629, 2010). In this paper we describe how to construct and use a microfluidic chip to study the response of single yeast cells to dynamic perturbations of their fluid environment. The method involves creation of a photoresist master mold followed by subsequent creation of a polydimethylsiloxane (PDMS) microfluidic chip for maintaining live yeast cells in a channel with two inputs for stimulating the cells. We emphasize simplicity and the methods discussed here are accessible to the average biological laboratory. We cover the basic toolbox for making microfluidic lab-on-a-chip devices, and the techniques discussed serve as a starting point for creating sophisticated microfluidic devices capable of implementing more complicated experimental protocols.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Análise de Célula Única/instrumentação , Leveduras/citologia , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Dimetilpolisiloxanos/química , Técnicas Analíticas Microfluídicas/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Microtecnologia/instrumentação , Microtecnologia/métodos , Análise de Célula Única/métodosRESUMO
All cells perceive and respond to environmental stresses through elaborate stress-sensing networks. Yeast cells sense stress through diverse signaling pathways that converge on the transcription factors Msn2 and Msn4, which respond by initiating rapid, idiosyncratic cycles into and out of the nucleus. To understand the role of Msn2/4 nuclear localization dynamics, we combined time-lapse studies of Msn2-GFP localization in living cells with computational modeling of stress-sensing signaling networks. We find that several signaling pathways, including Ras/protein kinase A, AMP-activated kinase, the high-osmolarity response mitogen-activated protein kinase pathway, and protein phosphatase 1, regulate activation of Msn2 in distinct ways in response to different stresses. Moreover, we find that bursts of nuclear localization elicit a more robust transcriptional response than does sustained nuclear localization. Using stochastic modeling, we reproduce in silico the responses of Msn2 to different stresses, and demonstrate that bursts of localization arise from noise in the signaling pathways amplified by the small number of Msn2 molecules in the cell. This noise imparts diverse behaviors to genetically identical cells, allowing cell populations to "hedge their bets" in responding to an uncertain future, and to balance growth and survival in an unpredictable environment.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/genética , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Algoritmos , Núcleo Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Redes Reguladoras de Genes , Glucose/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Microscopia de Fluorescência , Modelos Biológicos , Modelos Genéticos , Mutação , Nitrogênio/farmacologia , Concentração Osmolar , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/fisiologia , Sorbitol/farmacologia , Estresse Fisiológico/fisiologia , Imagem com Lapso de Tempo , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
We describe the development and characterization of a system that allows the rapid and specific induction of individual genes in the yeast Saccharomyces cerevisiae without changes in nutrients or temperature. The system is based on the chimeric transcriptional activator Gal4dbd.ER.VP16 (GEV). Upon addition of the hormone ß-estradiol, cytoplasmic GEV localizes to the nucleus and binds to promoters containing Gal4p consensus binding sequences to activate transcription. With galactokinase Gal1p and transcriptional activator Gal4p absent, the system is fast-acting, resulting in readily detectable transcription within 5 min after addition of the inducer. ß-Estradiol is nearly a gratuitous inducer, as indicated by genome-wide profiling that shows unintended induction (by GEV) of only a few dozen genes. Response to inducer is graded: intermediate concentrations of inducer result in production of intermediate levels of product protein in all cells. We present data illustrating several applications of this system, including a modification of the regulated degron method, which allows rapid and specific degradation of a specific protein upon addition of ß-estradiol. These gene induction and protein degradation systems provide important tools for studying the dynamics and functional relationships of genes and their respective regulatory networks.