FRET imaging of protein-protein interactions inside living cells

National Institute On Alcohol Abuse And Alcoholism

Investigators

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Abstract

The principal aim of the Section on Cellular Biophotonics is to use imaging techniques, such as two-photon microscopy, spectral imaging, fluorescence lifetime microscopy, 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. Our Sections initial efforts concentrated on 1. Building and testing a laser-scanning microscope specifically designed for studying protein-protein interactions in living cells, 2. Develop new methods for measuring FRET, and 3. Overcoming some of the practical limitations of FRET imaging. The microscope we have constructed is a fully functional laser scanning two-photon microscope, with the additional capabilities of measuring florescent emission spectra (spectral imaging), fluorescent lifetime decays (FLIM), and fluorescent anisotropy lifetime decays (rFLIM). These added capabilities make it specifically useful for monitoring FRET between either dissimilar (Hetero-FRET) or similar (Homo-FRET) fluorophores. We have also produced a set of FRET standards in our section and have provided these FRET standards to over 90 research groups including laboratories in Germany, Spain, Canada, Netherlands, Austria, Singapore, Switzerland, and Denmark. Currently we have 4 working projects in the lab. The first project is involved in concluding our feasibility and methodological studies on FRET imaging. The last four projects initiate the next phase in our Sections activities where our microscopes unique capabilities are utilized to address biological questions: 1. We have generated Homo-FRET reference standards that will be used in interpreting anisotropy decay experiments. This relatively new method has the potential for monitoring how proteins form multimeric structures, and their stoichiometry in living cells. 2. The second project has been following up on an observation we have made where we measure more FRET between GFP type fluorophores than are predicted by FRET theory. While we do not understand the mechanism of this additional energy transfer, our working hypothesis is that because GFP fluorophores are protected from the external environment by its beta-barrel protein structure, vibrational dequenching of excited states are slowed and thus allow for coherent energy transfer between fluorescent proteins. If correct, this would be one of the first examples of a biology adaptation to exploit quantum mechanical behavior at room temperature. This phenomenon might also be useful for generating components required for building quantum computers. 3. Our third project uses anisotropy lifetime decay analysis to monitor changes in the multimeric structure of Cam kinase-II. This abundant synaptic enzyme has been shown to play a pivotal role in learning and memory. It is believed that long-lived structural changes in this protein complex might be the embodiment of some forms of memory. Our results indicate that structural changes associated with Cam kinase-II activation can be detected with anisotropy imaging. 4. Finally, the Jain foundation has generously established a gift fund in support of our laboratories investigations into the functions of Dysferlin, a protein that is known to be responsible for LGMD2B/Miyoshi muscular dystrophy. Our experiments indicated that anti-sense morpholinos against Dysferlin injected into developing sea urchin embryos inhibit cell division and wound healing. Accordingly, we expanded this study to investigate the role of Dysferlin in calcium signaling. We have found that upon wounding, cells depolarize and secrete ATP by a mechanism involving agatoxin sensitive voltage-gated calcium channels. Neighboring cells respond to the secreted ATP by a mechanism thought to involve P2X receptors as well as voltage-gated calcium channels. Anti-sense morpholinos against Dysferlin block the secretion of ATP supporting the hypothesis that Dysferlin might act by mediating calcium triggered exocytosis.