RESUMO
The nanoscale arrangement of ligands can have a major effect on the activation of membrane receptor proteins and thus cellular communication mechanisms. Here we report on the technological development and use of tailored DNA origami-based molecular rulers to fabricate "Multiscale Origami Structures As Interface for Cells" (MOSAIC), to enable the systematic investigation of the effect of the nanoscale spacing of epidermal growth factor (EGF) ligands on the activation of the EGF receptor (EGFR). MOSAIC-based analyses revealed that EGF distances of about 30-40 nm led to the highest response in EGFR activation of adherent MCF7 and Hela cells. Our study emphasizes the significance of DNA-based platforms for the detailed investigation of the molecular mechanisms of cellular signaling cascades.
Assuntos
Fator de Crescimento Epidérmico , Receptores ErbB , Humanos , DNA/química , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Células HeLa , Ligantes , Transdução de SinaisRESUMO
In vitro cell-based experiments are particularly important in fundamental biological research. Microscopy-based readouts to identify cellular changes in response to various stimuli are a popular choice, but gene expression analysis is essential to delineate the underlying molecular dynamics in cells. However, cell-based experiments often suffer from interexperimental variation, especially while using different readout methods. Therefore, establishment of platforms that allow for cell screening, along with parallel investigations of morphological features, as well as gene expression levels, is crucial. The droplet microarray (DMA) platform enables cell screening in hundreds of nanoliter droplets. In this study, a "Cells-to-cDNA on Chip" method is developed enabling on-chip mRNA isolation from live cells and conversion to cDNA in individual droplets of 200 nL. This novel method works efficiently to obtain cDNA from different cell numbers, down to single cell per droplet. This is the first established miniaturized on-chip strategy that enables the entire course of cell screening, phenotypic microscopy-based assessments along with mRNA isolation and its conversion to cDNA for gene expression analysis by real-time PCR on an open DMA platform. The principle demonstrated in this study sets a beginning for myriad of possible applications to obtain detailed information about the molecular dynamics in cultured cells.
Assuntos
DNA Complementar , Linhagem Celular , Expressão Gênica , Análise em Microsséries/métodos , RNA Mensageiro/genéticaRESUMO
Simple and rapid imaging and analysis of 2D and 3D cell culture compatible with miniaturized arrays of nanoliter droplets are essential for high-throughput screening and personalized medicine applications. In this study, we have developed a simple one-step, cost-effective and sensitive colorimetric method for the analysis of cell viability in 2D and 3D cell cultures on a nanoliter droplet microarray. The method utilizes a flatbed document scanner that detects a color change in response to cell metabolism in nanoliter droplets with high sensitivity in a single step without the need for expensive specialized equipment. This new nanoliter-based method is faster and more sensitive than equivalent methods using multi-well plate assays. The method detects quantifiable signal from as few as 10 cells and requires only 5 min. This is 2.5 to 10-fold more sensitive and 12 times faster than the same assay in multi-well plates. The method is simple, affordable, fast and sensitive. It can be used for various applications including high-throughput cell-based and biochemical screenings.