Team:Czech Republic/Microfluidics

Signal transduction characterisation on-chip

Example filling of microfluidic channel.

We developed a new fluorescence reporter assay system on-chip which allows dynamic detection of GPCR signaling in yeast using live fluorescence microscopy. First, fluorescent reporter proteins are connected to specific functional products signalling activation of the specific G protein–coupled receptor (GPCR). Details on the synthesis and function of reporter plasmids are provided by Module 1. Synthesised reporter plasmids were transformed to specific yeast strains to enable characterisation of yeast signalling using set of orthogonal signals designed and synthesised by Module 2.

Fabricated microfluidic devices were used to characterise the signal transduction between selected yeast strains. Two cell types are selected for each experiment. One cell type transmits the signal in the form of specific pheromone molecules. And the other cell type produces the Green Fluorescent Protein (GFP) when the signal is received if the pheromone molecules fit the specific receptor. Both cell types are introduced to the microfluidic device in parallel through separated inlets. Due to the laminar nature of the flow, the individual cell types remain spatially separated inside the microfluidic device. The spatial separation is well visible during the filling of the microfluidic device. The flow is stopped when the filling of the microfluidic device is finished, and the rest of the experiment is performed statically. Subsequently, time lapse fluorescence microscopy is used to dynamically measure levels of GFP expression in the cells that receive the signal.

Level of fluorescence and its dependence on the distance from transmitting cells. Performed on S. cerevisiae wildtype pheromone signalling molecules and receptors.

All experiments were conducted on Saccharomyces cerevisiae cells. Signal transduction was tested between the following types:

  • POSITIVE CONTROL: Wildtype MATx ↝ MATa: STE2 receptor from Sacharomyces cerevisiae
  • POSITIVE CONTROL: MATa expressing "Candida parapsilosis" alpha-factor; \(\Delta\)Bar ↝ MATa: STE2 receptor from Candida parapsilosis
  • NEGATIVE CONTROL: MATa expressing "Candida parapsilosis" alpha-factor; \(\Delta\)Bar ↝ MATa: STE2 receptor from Sacharomyces cerevisiae

Results of the experiment show...