FRET imaging of protein-protein interactions inside living cells

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The principal aim of the Section on Cellular Biophotonics is to use imaging and spectroscopy techniques, such as two-photon microscopy, spectral imaging, fluorescence lifetime microscopy, fluorescence correlation spectroscopy (FCS), and fluorescence anisotropy analysis to study how protein complexes regulate synaptic function in living cells. Recently, we have concentrated our efforts on utilizing Forster Resonance Energy Transfer (FRET) to monitor protein-protein interactions. This method has great potential for studying protein interactions because it is sensitive to changes in the distance separating two fluorophores on the 1-10 nm scale. FRET imaging in conjunction with the development of spectral variants of Green Fluorescent Protein (GFP) provides the opportunity to genetically tag synaptic proteins of interest and monitor their interactions with other labeled proteins in real time. Currently we have 5 projects in the lab: The goal of the first project is to adapt FPFA (fluorescence polarization and fluctuation analysis), a mixture of anisotropy lifetime decay analysis and FCS, to: monitoring dynamic changes in protein complex structure, to identify specific sites of protein-protein interactions, and to automate FPFA to enable the wide-scale biophysical analysis of protein-protein interactions between hundreds of potentially interacting proteins that comprise the interactome (a cells complete network of molecular interactions). Our second project is aimed at applying FPFA in collaborations with Drs. Anne Kenworthy and Eric Long. The third project is involved in developing fiber-optics based spectroscopy for use in monitoring fluorescent probes expresses in living mice. Once perfected, these fiber-based systems will be used to monitor specific changes in fluorophores expressed in specific subsets of neurons of transgenic mice as they perform behavioral tasks. Our biological goal in this third project is to understand how the basal ganglia controls voluntary action at the molecular level. Our technological goal is to adapt fiber optics and advanced spectroscopic techniques to dynamically monitor bio-sensors and/or protein-protein interaction in neurons deep within the functioning brain. Our fourth project is aimed at understanding the mechanism of recently discovered ultra-fast energy transfer observed in assemblies of fluorescent proteins. Our fifth project, in collaboration with Drs. Paul Blank and Wieb van der Meer, is to develop the underlying theory to allow accurate estimates of distance between biological components based on FRET measurements despite uncertainties in fluorophore orientation.