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LMP - Section on Cellular Biophotonics (CBP)
Steven Vogel, PhD, Acting Chief
National Institute on Alcohol Abuse and Alcoholism
National Institutes of Health
5625 Fishers Lane, Room TS-06F:MSC 9411
Bethesda MD 20892-9413
telephone: +1 301.496.9288
fax: +1 301.480.0466
The mission of the Section on Cellular Biophotonics, of the Laboratory of Molecular Physiology (SCB/LMP), NIAAA, is to determine how protein complexes are formed and maintained in living cells, and how they participate in mediating or regulating cellular functions, primarily using imaging and spectroscopic techniques. We are particularly interested in protein complexes that respond to an influx in calcium to regulate synaptic function.
FRET imaging of protein-protein interactions in living cells
The Section on Cellular Biophotonics uses 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 Förster’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’s initial efforts concentrated on 1. Building and testing a laser scanning microscope specifically designed for studying protein-protein interactions in living cells, 2. Developing 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.
Currently we are working on 2 major projects:
Our first project uses time-resolved fluorescence anisotropy 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. We are currently interested in determining how the CaMKII holoenzyme is assembled from a and subunits. The specific combination of these subunits is thought to impact CaMKII’s ability to regulate synaptic efficacy. We are also interested in exploiting a FRET-based assay for CaMKII activation that we have recently developed to determine what factors regulate the time-course of kinase activation in dendritic spines.
The second project, is investigating the role of Dysferlin in wound repair, and has direct relevance to understanding the molecular basis of Limb Girdle and Miyoshi Muscular Dystrophy. Both of these syndromes are caused by mutations in the protein Dysferlin, but the function of Dysferlin itself is not known. Our working hypothesis is that Dysferlin mediates calcium triggered membrane fusion utilized by plasma membrane wound repair mechanisms. This project combines our laboratory’s long-held interest and expertise in using live cell imaging to study the mechanism of calcium triggered exocytosis in sea urchin eggs, with our more recent work using laser wounding and two-photon imaging to examine plasma membrane repair mechanisms. The Dysferlin project is funded, in part, by a gift from the Jain Foundation.
Covian-Nares JF, Smith RM, Vogel SS. Two independent forms of compensatory endocytosis maintain embryonic cell surface homeostasis during early development.
Dev Biol 316: 135-48, 2008.
Koushik SV, Vogel SS. Energy migration alters the fluorescence lifetime of Cerulean: Implications for FLIM-FRET measurements.
J Biomed Optics 13:03 1204, 2008.
Key prior publication:
Vogel SS, Thaler C, Koushik SV. Fanciful FRET.
Science STKE 2006(331)re2.