The mating pathway in Saccharomyces cerevisiae has been the focus of considerable research effort, yet many quantitative aspects of its regulation still remain unknown. Using an integrated approach involving experiments in microfluidic chips and computational modelling, we studied gene expression and phenotypic changes associated with the mating response under well-defined pheromone gradients. Here we report a combination of switch-like and graded pathway responses leading to stochastic phenotype determination in a specific range of pheromone concentrations. Furthermore, we show that these responses are critically dependent on mitogen-activated protein kinase (MAPK)-mediated regulation of the activity of the pheromone-response-specific transcription factor, Ste12, as well as on the autoregulatory feedback of Ste12. In particular, both the switch-like characteristics and sensitivity of gene expression in shmooing cells to pheromone concentration were significantly diminished in cells lacking Kss1, one of the MAP kinases activated in the mating pathway. In addition, the dynamic range of gradient sensing of Kss1-deficient cells was reduced compared with wild type. We thus provide unsuspected functional significance for this kinase in regulation of the mating response.

We demonstrate microscopic fluidic control and memory elements through the use of an aqueous viscoelastic polymer solution as a working fluid. By exploiting the fluid's non-Newtonian rheological properties, we were able to demonstrate both a flux stabilizer and a bistable flip-flop memory. These circuit elements are analogous to their solid-state electronic counterparts and could be used as components of control systems for integrated microfluidic devices. Such miniaturized fluidic circuits are insensitive to electromagnetic interference and may also find medical applications for implanted drug-delivery devices.

Bacteria and yeast frequently exist as populations capable of reaching extremely high cell densities. With conventional culturing techniques, however, cell proliferation and ultimate density are limited by depletion of nutrients and accumulation of metabolites in the medium. Here we describe design and operation of microfabricated elastomer chips, in which chemostatic conditions are maintained for bacterial and yeast colonies growing in an array of shallow microscopic chambers. Walls of the chambers are impassable for the cells, but allow diffusion of chemicals. Thus, the chemical contents of the chambers are maintained virtually identical to those of the nearby channels with continuous flowthrough of a dynamically defined medium. We demonstrate growth of cell cultures to densely packed ensembles that proceeds exponentially in a temperature-dependent fashion, and we use the devices to monitor colony growth from a single cell and to analyze the cell response to an exogenously added autoinducer.

Adaptation in signaling systems, during which the output returns to a fixed baseline after a change in the input, often involves negative feedback loops and plays a crucial role in eukaryotic chemotaxis. We determined the dynamical response to a uniform change in chemoattractant concentration of a eukaryotic chemotaxis pathway immediately downstream from G protein–coupled receptors. The response of an activated Ras showed near-perfect adaptation, leading us to attempt to fit the results using mathematical models for the two possible simple network topologies that can provide perfect adaptation. Only the incoherent feedforward network accurately described the experimental results. This analysis revealed that adaptation in this Ras pathway is achieved through the proportional activation of upstream components and not through negative feedback loops. Furthermore, these results are consistent with a local excitation, global inhibition mechanism for gradient sensing, possibly with a Ras guanosine triphosphatase–activating protein acting as a global inhibitor.