The unique features of fluorescence fluctuation spectroscopy (FFS) make this technique attractive for cellular applications. FFS requires no external perturbation to determine kinetic and molecular properties with submicron spatial resolution and single molecule sensitivity. Especially, the application of FFS to proteins tagged with green fluorescent protein (GFP) has the potential to revolutionize in vivo studies. The long-term objective of the proposed research lies in the concurrent development and application of 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. Fluorescence correlation spectroscopy (FCS) uses the autocorrelation function to determine kinetic parameters, such as the diffusion coefficient. In addition to regular FCS two other fluctuation techniques, the photon counting histogram (PCH) and scanning FCS will be used. PCH, a recently developed technique, determines the molecular brightness, which will be used to address the association of proteins. Scanning FCS has the potential to detect immobilized proteins. Together, the fluctuation techniques provide complementary information necessary for a successful characterization of biochemical processes in vivo. To assess the true potential of these techniques in vivo a thorough characterization will be performed and the statistical accuracy of each technique will receive special emphasis. The properties of EGFP and the autofluorescence in the different cellular compartments will be characterized in order to understand the quantitative range of FFS measurements in living cells. The fluctuation techniques will be applied to study Retinoid X Receptor (RXR), a nuclear receptor, in vivo. Nuclear receptors are ligand controlled transcription regulators and RXR has been identified as the key regulator of hormone receptors. The oligomerization state of RXR, which ranges from tetramer to monomer, serves as a control mechanism for its activation. The resolution of the oligomenzation state of RXR by fluctuation techniques will be at the focus of this in vivo study. In addition, the binding of RXR to DNA will be studied.
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