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. Currently we have 2 projects in the lab. The first project is involved in building a two-photon microscope specifically designed to study protein complexes in living cells. The microscope we are assembling will be capable of simultaneously measuring time resolved fluorescence anisotropy, and the fluctuations in fluorescence intensity that can then be analyzed by fluorescence correlation spectroscopy (FCS). Our second project uses anisotropy lifetime decay analysis and FCS analysis to monitor changes in the multimeric structure of Cam kinase-II. In the brain this abundant synaptic enzyme is thought to be a calcium spike frequency detector, and has been shown to play a pivotal role in learning and memory. In the heart CaMKII activity has been linked to several forms of heart disease. Our results indicate that structural changes associated with CaM kinase-II activation can be detected using anisotropy imaging and FCS,and we now wish to image the activation of this protein complex in living zebrafish hearts.

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National Institute on Alcohol Abuse and Alcoholism
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Nguyen, Tuan A; Puhl 3rd, Henry L; Pham, An K et al. (2018) Auto-FPFA: An Automated Microscope for Characterizing Genetically Encoded Biosensors. Sci Rep 8:7374
Sarkar, Pabak; Davis, Kaitlin A; Puhl 3rd, Henry L et al. (2017) Deciphering CaMKII Multimerization Using Fluorescence Correlation Spectroscopy and Homo-FRET Analysis. Biophys J 112:1270-1281
Kumar, Santosh; Rajagopalan, Sumati; Sarkar, Pabak et al. (2016) Zinc-Induced Polymerization of Killer-Cell Ig-like Receptor into Filaments Promotes Its Inhibitory Function at Cytotoxic Immunological Synapses. Mol Cell 62:21-33
Nguyen, Tuan A; Sarkar, Pabak; Veetil, Jithesh V et al. (2015) Covert Changes in CaMKII Holoenzyme Structure Identified for Activation and Subsequent Interactions. Biophys J 108:2158-70
Kumar, Santosh; Sarkar, Pabak; Sim, Malcolm J W et al. (2015) A single amino acid change in inhibitory killer cell Ig-like receptor results in constitutive receptor self-association and phosphorylation. J Immunol 194:817-26
Kraft, Lewis J; Nguyen, Tuan A; Vogel, Steven S et al. (2014) Size, stoichiometry, and organization of soluble LC3-associated complexes. Autophagy 10:861-77
Vogel, Steven S; van der Meer, B Wieb; Blank, Paul S (2014) Estimating the distance separating fluorescent protein FRET pairs. Methods 66:131-8
Cui, Guohong; Jun, Sang Beom; Jin, Xin et al. (2014) Deep brain optical measurements of cell type-specific neural activity in behaving mice. Nat Protoc 9:1213-28
Cui, Guohong; Jun, Sang Beom; Jin, Xin et al. (2013) Concurrent activation of striatal direct and indirect pathways during action initiation. Nature 494:238-42
Kerr, Jaclyn P; Ziman, Andrew P; Mueller, Amber L et al. (2013) Dysferlin stabilizes stress-induced Ca2+ signaling in the transverse tubule membrane. Proc Natl Acad Sci U S A 110:20831-6

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