A systematic approach is used to study dynamics in the RAS/cAMP/PKA pathway in budding yeast by combining fluorescence microscopy with image analysis and systems biology. The temporal and spatial information gained from in vivo fluorescence microscopy experiments is excellent for studying protein expression, dynamics and protein-protein interactions on a cell to cell basis, which provides input data to build up mathematical models and networks.
The RAS/cAMP/PKA pathway serves as an important regulator of the metabolic and transcriptional activity of yeast cells and is partially well characterized. However, the regulatory feedback loop, transferring information from PKA to RAS, remains unclear. In our first approach we are going to perturb the signal flow using genetic and chemical methods, and observe resulting changes in GFP-tagged Msn2p localization, which indicates PKA activity.
For validation purposes we study the pathway components and deletion mutations under their native promoters. We here show how the activation state of the system changes upon removal of some of the key components, e.g. Dtpk1, Dbcy1. Following the localization dynamics of Msn2p over time under both different genetic (internal) modifications and under different stress factors, such as light or heat, we detect different localization patterns. For instance, no apparent oscillation of Msn2p between the nuclei and the cytosol can be detected for Dbcy1, a phenomenon that is present for other deletion mutants.
Introduced signal disruptions can lead to clarification of potential interactions between pathway elements. Furthermore, possible physical interactions between proteins are planned to be confirmed by using FRET-FLIM. Continuous oscillations in a biological system are signs that there are one or several feedback loops. The current observations indicate that oscillations in Msn2p localization are driven by one or more feedback mechanisms in the RAS/cAMP/PKA pathway, since modifications in probable feedback components result in changed Msn2p localization patterns.
AUTHORS AND AFFILIATIONS
Katarina Logg, Dept. of Applied Physics, Chalmers University
Kristofer Bodvard, Dept. of Applied Physics, Chalmers University
Mikael Käll, Dept. of Applied Physics, Chalmers University
Piotr Sliwa, Dept. of Cell & Molecular Biology, Gothenburg University
Anders Blomberg, Dept. of Cell & Molecular Biology, Gothenburg University
Mats Jirstrand, Fraunhofer-Chalmers Centre
Mats Kvarnström, Fraunhofer-Chalmers Centre
Joachim Almquist, Fraunhofer-Chalmers Centre