| Systems biology has grown immensely in the wake of human genome project. In recent years, there has been a tremendous increase in measurement capabilities (e.g., microarray and proteomic technologies, improved reporter genes). However, future success depends not only on effective measurement techniques but also on the design and implementation of appropriate experimental stimuli. In this project, we investigate experimental approaches where the long-term dynamics of single cells subjected to a dynamic environment can be observed. We use microfluidic technology to develop a device where cells can be subjected to a stable and precise chemical gradient. We over- come the typical problem with many earlier gradient devices, where the high fluid flow needed to maintain the gradient renders such devices undesirable for the study of yeast cells. We use the gradient device along with fluorescence microscopy and molecular biology techniques to study gradient sensing and cell polarization during mating in the model organism Saccharomyces cerevisiae. We generalize the chemical gradient device such that the direction of the gradient can be specified as a function of time. The response of yeast cells to spatiotemporal signals generated by this device reveals aspects of yeast polarization adaptation that are unlikely to be observed in static environments. An integrated computational and experimental analysis of the pheromone response of yeast will provide a detailed understanding of the gradient sensing in yeast. Because MAP kinase signaling cascades and cell polarization machinery are conserved in most eukaryotes, understanding of the pheromone pathway should lead to improved models of cell polarization and gradient sensing in more complex organisms. |
