IN VIVO FLUORESCENCE FLUCTUATION SPECTROSCOPY

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DESCRIPTION (provided by applicant): The unique features of fluorescence fluctuation spectroscopy (FFS) make this technique attractive for cellular applications. FFS determines kinetic and molecular properties of proteins with submicron resolution and single molecule sensitivity. Especially, the application of FFS to proteins tagged with a variant of green fluorescent protein has the potential to revolutionize in vivo studies. We will focus here on dual-color photon counting histogram (PCH) analysis. PCH is a relatively new FFS technique with the potential to provide quantitative information about protein association in living cells. Dual-color PCH exploits two fluorescent protein labels with distinctively different emission spectra. Separating the two colors into different detection channels strongly increases the sensitivity of PCH in detecting protein association. We will adept dual-color PCH for in vivo studies, implement global analysis methods and thoroughly characterize the technique. 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 PCH will be applied to study the interactions between nuclear receptors and their coregulators in vivo. Nuclear receptors are transcription factors that turn gene expression on and off. They inhibit or enhance transcription by recruiting an array of coactivat6r or corepressor proteins. The quantitative characterization of the oligomerization state of the receptorcoregulator complex by dual-color PCH will be at the focus of this study. Nuclear receptors affect processes as diverse as reproduction, development, and general metabolism, and have been implicated in cancer, diabetes, and many other diseases. Dual-color PCH could help in fighting these diseases by providing detailed information about the protein interactions that govern gene expression in cells.