Dynamics of molecular interactions in live cells

Molecular interactions such as binding reactions of proteins belong to the most important mechanisms for the regulation of biological processes. The majority of elementary biological interactions have been discovered by screening approaches and were then characterized in vitro outside of intact cells to determine biophysical parameters such as affinities, as well as binding or dissociation rates. We believe that these interaction parameters do not represent the conditions of live cells, with their complex micro-environment of bio-molecules. We hypothesize that crucial biological processes are regulated by alterations of affinities and interactions and that these dynamic binding and dissociation processes should be studied in intact cells, if we want to understand them thoroughly. We developed a method, which allows measuring the dynamics of protein interactions, as well as the half-lives of protein complexes in live cells by combining two established laser microscopy techniques. In the course of the proposed project, we intend to develop this method further and to apply it to physiological protein concentrations in order to characterize molecular interactions of a biological key process: the inflammatory signal transduction leading to activation of the transcription factor NF-kappa B. To that end, we want to exploit a new technology, which allows a specific modification of the genome of cells. This will enable us to couple fluorescent proteins as markers with the signaling molecules of interest without changing their expression levels and to apply our novel microscopy technique to proteins in their physiological context. Thereby, we want to investigate whether and to which extent molecular interactions or compositions of signaling complexes change in the course of signaling. Furthermore, we want to study, whether interaction parameters are different in distinct compartments of the cell such as nucleus and cytosol. Finally, we want to elucidate, how competing interactions of signaling molecules are balanced during inflammation, which might also result in new strategies to develop substances targeting these interaction networks. 1

Physiological processes are crucially regulated by molecular interactions in particular by those between proteins that have signaling functions. However, these interactions are difficult to study in the natural environment within living cells - and have been investigated mostly by classical biochemical methods in test tubes (in vitro). We hypothesized that minor changes in binding affinities as well as molecular dynamics of interactions might cause shifts from one type of interaction to a different one within live cells. To address that we established novel methods that are based on a quantum physical phenomenon termed fluorescence resonance energy transfer (FRET), which can be measured by means of microscopy or cytometry in vivo. While focusing on molecular interactions of components of the NF-B signaling pathway, a central mediator of inflammation, the main aim of this project was to improve the analysis of molecular interactions by determining crucial biophysical parameters, such as interaction affinities or binding ratios within living cells. "Measure what is measurable and make measurable what is not so." This famous quote by Galileo Galilei was the guiding principle of this project. As starting point to investigate protein-protein interactions, we compared common co-localization analysis methods and developed a novel technique, which combined object recognition with statistical pixel-intensity correlation. Furthermore, we demonstrated that co-localization analysis alone can deliver false-positive indications of protein interactions, while the above-mentioned FRET effect, when properly measured, is a robust assessment of physical proximity and thus direct interaction. Next, we developed a novel FRET analysis technique, which allows the determination of the binding state of two proteins with a high degree of accuracy. A further fitting algorithm combined with the acquisition of large data sets, either by microscopy or flow cytometry, allowed the determination of robust measures of protein-protein interaction far beyond a mere qualitative assessment. Using this method, we studied changes in affinity between the two major subunits of the IKK complex, IKK1 and IKK2, revealing that the interaction is transiently decreased after activating the NF-B signaling cascade. This might be the basis for interactions between IKKs and other proteins, allowing a dynamic regulation of phosphorylation events during inflammatory signaling. In addition, we studied molecular links between the oncogene c-Myc, IKK1 and the coagulation factor F3, which provided deeper insights into correlations between cancer, inflammation and thrombosis. Taken together, our project is important for the scientific field as it developed novel methods for a quantitative assessment of protein interactions in living cells - and also because it elucidated some important parameters of protein-protein interactions for components of the inflammatory NF-B signaling pathway, as well as links between oncogenes, inflammatory molecules and coagulation factors.