Because of its unique features, fluorescence fluctuation spectroscopy (FFS) is an attractive technique for cellular applications. It determines kinetic and molecular properties of proteins with submicron resolution and single molecule sensitivity. Especially, the application of FFS to cellular proteins tagged with a fluorescent protein has the potential to provide quantitative information about their interactions in a living cell. We introduce dual-color multi-excitation FFS to quantify the homo- and hetero-interactions of two proteins labeled with distinct fluorescent colors. Dual-color multi-excitation FFS achieves the necessary sensitivity by exploiting the differences in the excitation and emission properties of the fluorescent proteins. We will develop dual-color multi excitation FFS for in vivo studies, implement global analysis methods and thoroughly characterize the technique. So far most FFS brightness experiments have been limited to the cell nucleus. We will extend the reach of FFS brightness analysis to the cytoplasm and to the plasma membrane by developing a technique that takes the cell shape into account. The long-term objective of the proposed research lies in the concurrent development and application of fluorescence fluctuation techniques, so that their full potential for in vivo studies is realized. The impact of this new technology will be felt in many biological areas with applications ranging from basic research in cell biology to pharmaceutical drug screening. Dual-color multi-excitation FFS will be applied to study the interactions between the nuclear receptor RXR and its coregulators SRC-1 in vivo. The quantitative characterization of the oligomerization state of the receptor-coregulator complex will be at the focus of this study. In addition, we characterize the oligomerization of dynamin and it interaction with endophilin both in the cytoplasm and on the plasma membrane. Nuclear receptors and dynamin are implicated in a number of diseases, such as cancer, diabetes, and neurodegenerative diseases. In vivo FFS studies could help in fighting these diseases by providing detailed information about the protein interactions and may lead to the identification of targets for drug development. The goal of the project is the development of a spectroscopic tool with the unique ability to quantify protein interactions directly inside a living cell. Knowledge of protein interactions helps to identify the molecular cause or mechanism underlying a disease. It also provides information that may aid in the development of therapies.
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