A microfluidic study on the regulation of ribosome biogenesis in saccharomyces cerevisiae

Abstract

Microfluidic bioreactors are economical, efficient and usable reactors for conducting biological studies aiming to observe cells under continuous flow. In the present work, microchips from COP (cyclo olefin copolymer) material, which has high fluorescence permeability, were produced for the intended experiments. Entrance hole drilling, hot embossing and thermocompression bonding process steps and the related parameters were optimized throughout the chip production. Two simultaneous experiments, each of which is repeated eight times by means of 16 independent chambers of a single microbioreactor were conducted under steady state. The variation of cells marked with NOP56 red fluorescence during the treatment of hydroxyurea- ribonucleotidreductase inhibitor- was analyzed in the microbioreactor for 16 hours. In order to investigate the effect of various concentrations of hydroxymethylfurfural, the cell growth inhibitor on ribosome biogenesis, the same type of cells were followed in the microbioreactor using YNB medium during simultaneous experiments. The effects of metformin, the inhibitor of the rapamycin protein complex in mammals, on cell growth and ribosome biogenesis were also examined in NOP56:RFP tagged yeast cells. In these experiments, the yeast cells were periodically observed under the Nikon TI-E inverse microscope, and brightfield and fluorescence images were taken, and images taken were processed using the Fiji-ImageJ program. Based on the obtained and calculated data, the time profiles of cell area, circumference, volume and integrated fluorescence intensity were extracted and interpreted as the cells, response to the inhibitors. As a result of single cell processing of fluorescence images, hydroxymethylfurfural, hydroxyurea and metformin inhibitors suppressed the expression of NOP56 gene. In the experiments containing hydroxymethylfurfural and hydroxyurea inhibitors, it was observed that the cells recovered in 10 hours when they were fed with fresh medium.