Abstract
We report on the spatial and temporal signaling properties of a yeast pheromone-based cell communication and amplifier system. It utilizes the Saccharomyces cerevisiae mating response pathway and relies on diffusion of the pheromone α–factor as key signaling molecule between two cell types. One cell type represents the α–factor secreting sensor part and the other the reporter part emitting fluorescence upon activation. Although multi-cellular signaling systems promise higher specificity and modularity, the complex interaction of the cells makes prediction of sensor performance difficult. To test the maximum distance and response time between sensor and reporter cells, the two cell types were spatially separated in defined compartments of agarose hydrogel (5 × 5 mm) and reconnected by diffusion of the yeast pheromone. Different ratios of sensor to reporter cells were tested to evaluate the minimum amount of sensor cells required for signal transduction. Even the smallest ratio, one α–factor-secreting cell to twenty reporter cells, generated a distinct fluorescence signal. When using a 1:1 ratio, the secreted pheromone induced fluorescence in a distance of up to four millimeters after six hours. We conclude from both our experimental results and a mathematical diffusion model that in our approach: (1) the maximum dimension of separated compartments should not exceed five millimeters in gradient direction; and (2) the time-limiting step is not diffusion of the signaling molecule but production of the reporter protein.
Highlights
In the past years, advancements in the development of microbial biosensors promised technical progress to detect biologically available compounds or biohazards
Yeast sensor cells feature an inducible or repressible promoter element that controls the expression of a reporter gene such as enhanced green fluorescent protein (EGFP), resulting in ―lights on‖
In order to analyze this system in more detail, we examined α–factor diffusion as well as mating response and fluorescence induction in reporter cells
Summary
Advancements in the development of microbial biosensors promised technical progress to detect biologically available compounds or biohazards. Respective yeast sensor cells were reported for analytes like heavy metals [4], organic compounds [5] and hormone active substances [6,7] All of these systems are based on a single yeast strain that senses the analyte, which drives expression of a marker gene. The recently described bimodular amplifier system [8] can be regarded as an extension of a unicellular sensor approach, in which the two functions—sensing of an analyte as the input signal and production of fluorescence as the output—are separated and connected by α–factor signaling Particular advantages of this multi-step system are signal amplification and modularization. In order to analyze this system in more detail, we examined α–factor diffusion as well as mating response and fluorescence induction in reporter cells To this end, sources of α–factor (synthetic or cell-secreted) and pheromone-responsive reporter cells were separately immobilized in 3D compartments based on agarose hydrogel. We report on the diffusion of α–factor in agarose hydrogel and its time- and space-dependent induction of spatially separated fluorescent S. cerevisiae reporter cells
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