FRET imaging of protein-protein interactions inside livi

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The principle 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?s 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 Section is comprised of Drs. Steven Vogel (Chief), Srinagesh Koushik (Research Fellow), Christopher Thaler (Postdoctoral IRTA), Jose Fernando Covian-Nares (Visiting Fellow), and Mr. Michael Pham (Post-baccalaureate IRTA). 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 used this microscope to develop a new photon efficient method for measuring FRET in living cells based on spectral imaging (Thaler et al. 2005. Biophys J 89:2736-2749). A manuscript, evaluating how quantitative spectral imaging is for discriminating proteins labeled with either blue (CFP) or yellow (YFP) variants of GFP has been published (Thaler, and Vogel. 2006. Cytometry A 69:904-911). We have identified several important issues that hinders the utilization of FRET imaging (Vogel et. al. 2006. Sci STKE 2006:re2) and have developed FRET imaging standards to help overcome one of these major obstacles (Koushik et al. 2006. manuscript revision submitted ). In collaboration with Dr. Ikedea?s Section we have optimized a method for measuring FRET using sensitized emission (Chen et. al. 2006. Biophys J 91:L39-41), and have validated the use of CFP as a FRET donor in conjunction with acceptor photo-bleaching (Thaler et. al. 2006. Nat Methods 3:491). Currently we have 5 working projects in the lab. The first two are involved in concluding our feasibility and methodological studies on FRET imaging. The last three 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 methodological project has been investigating how FRET efficiencies change when multiple acceptors are present. This type of problem is important, because many proteins form multimeric complexes, and the assembly and disassembly of these complexes might play an important role in regulating cell functions. The accepted mathematical formalism for dealing with this class of FRET problems assumes that the rates of energy transfer (to single acceptors) sum linearly when multiple acceptors are present. Our preliminary data suggests that this treatment underestimates the true FRET efficiency. We have developed a new theory for dealing with these problems based on probability theory, and we are currently testing this new formalism. 3. The third project, in collaboration with Dr. David Lovinger?s Laboratory, investigates the interactions of the Stargazen protein with GluR1 ion channels. GluR1 has been shown to play a decisive role in synaptic efficacy, as activity causes GluR1 to migrate to dendritic spines in hippocampal CA1 neurons. Stargazin, a member of the transmembrane AMPA receptor regulatory protein family has also been shown to affect the trafficking of GluR1 as well as its kinetics. We are using spectral analysis, FLIM analysis, and anisotropy analysis to investigate the interactions of stargazing with GluR1, as well as their multimeric structure. 4. The fourth project uses anisotropy lifetime decay analysis to monitor changes in the multimeric structure of Cam kinase-II. This abundant post-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. Preliminary results indicate that structural changes associated with Cam kinase-II activation can be detected with FRET imaging. 5. In our fifth project we are using two-photon microscopy to investigating the roles of Src kinase (an oncogene) and Dynamin (a protein that assembles into a coiled structure and is directly involved in membrane scission ) in regulating endocytosis and cell division. We have developed a new imaging based assay for testing the effects of either over expression of exogenous proteins, or down regulation of endogenous proteins, on compensatory endocytosis and cell division in developing sea urchin embryos. Using this assay we have found a surprising connection between endocytosis and cell division. Over expression of Src kinase, inhibits compensatory endocytosis and blocks cell division. Inhibitors of tyrosine phosphatase had similar effects, suggesting that the balance of tyrosine kinase and phosphatase activity plays a key role in how cells regulate endocytosis and cell division. Injection of an anti-sense morpholino against Dynamin also blocked endocytosis and cell division, and treatment with a peptide that blocks Dynamin?s interactions with other proteins through its SH3 (Src homology 3) domain also blocked endocytosis and cell division. Injection of an anti-sense morpholino against Src inhibited cell division, but did not inhibit endocytosis. These experiments suggest that dynamin-dependent endocytosis is required for cell division.