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 Forsters 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), and Jose Fernando Covian-Nares (Visiting Fellow). ? 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. Since our last report we have published two new manuscripts. Additionally we currently have a book chapter in press, and a manuscript under review. We have also produced a set of FRET standards in our section and have provided these FRET standards to over 60 research groups including laboratories in Germany, Spain, Canada, Netherlands, Austria, Singapore, Switzerland, and Denmark. ? ? Currently we have 5 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 and faster FRET between GFP type fluorophores than are predicted by FRET theory. After consulting with FRET experts from around the world, our current 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 FPs. If our hypothesis is 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. In our fourth project we are using two-photon microscopy to investigating the roles of 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. We have previously shown that 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 Dynamins 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.? ? 5. Finally, the Jain foundation has generously established a gift fund in support of our laboratories preliminary investigations into the functions of Dysferlin, a protein that is known to be responsible for LGMD2B/Miyoshi muscular dystrophy. Our preliminary 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.
| Sarkar, Pabak; Koushik, Srinagesh V; Vogel, Steven S et al. (2009) Photophysical properties of Cerulean and Venus fluorescent proteins. J Biomed Opt 14:034047 |
| Thaler, Christopher; Koushik, Srinagesh V; Puhl 3rd, Henry L et al. (2009) Structural rearrangement of CaMKIIalpha catalytic domains encodes activation. Proc Natl Acad Sci U S A 106:6369-74 |
| Thaler, Christopher; Vogel, Steven S (2006) Quantitative linear unmixing of CFP and YFP from spectral images acquired with two-photon excitation. Cytometry A 69:904-11 |
| Koushik, Srinagesh V; Chen, Huanmian; Thaler, Christopher et al. (2006) Cerulean, Venus, and VenusY67C FRET reference standards. Biophys J 91:L99-L101 |
| Chen, Huanmian; Puhl 3rd, Henry L; Koushik, Srinagesh V et al. (2006) Measurement of FRET efficiency and ratio of donor to acceptor concentration in living cells. Biophys J 91:L39-41 |
| Thaler, Christopher; Vogel, Steven S; Ikeda, Stephen R et al. (2006) Photobleaching of YFP does not produce a CFP-like species that affects FRET measurements. Nat Methods 3:491; author reply 492-3 |
| Vogel, Steven S; Thaler, Christopher; Koushik, Srinagesh V (2006) Fanciful FRET. Sci STKE 2006:re2 |
| Thaler, Christopher; Koushik, Srinagesh V; Blank, Paul S et al. (2005) Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer. Biophys J 89:2736-49 |
| Sen, Pabitra N; Basser, Peter J (2005) A model for diffusion in white matter in the brain. Biophys J 89:2927-38 |
| McNeil, Paul L; Miyake, Katsuya; Vogel, Steven S (2003) The endomembrane requirement for cell surface repair. Proc Natl Acad Sci U S A 100:4592-7 |
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