SP6 - Protein Network Dynamics

R. Pepperkok, Miriam Reiss  (EMBL)

Signaling and protein interaction networks in living cells are characterized by their spatial and temporal topology. Interactions are highly dynamic and can thus rapidly change place and intensity in response to external stimuli. In this way cells quickly respond to environmental changes necessary to re-program the cell for its altered challenges. Therefore, a proper understanding of the physiology and pathophysiology of cells and organisms requires a quantitative understanding of the spatial and temporal topology of interaction networks. Along these lines, we aim at the integration of the functional and biochemical information in into a living cell context. To this end, high-throughput microscopy methods have been developed and are used to generate first an interaction map of putative network components in living cells and subsequently determine its dynamics in response to external stimuli (Neumann et al., 2010).
We follow three major lines of research to address these challenges.

• Systematic analysis of localization and dynamics of the components of putative signaling and interaction networks predicted, identified ad GFP-tagged by network in different breast cancer cells in response to growth factor stimulation.
• Development of a high throughput fluorescence cross correlation platform (FCCS-P) to quantify protein interactions in living cells.
• Application of the FCCS-P to the generation of interaction maps and their dynamics for components of breast cancer relevant signaling and interaction networks.

Studying signaling at the single cell level. HeLa cells were activated with TNFapha (at the time point indicated with a yellow star) and then the nuclear localization of p38 protein was followed over time. While translocation of p38 into the nucleus is a fast event in the cell on the left, the cell on the right shows a delayed on-set of nuclear import. Hence, single cell resolution is mandatory to analyze this kind of events.