Significance Light microscopy advanced our understanding of cellular structure and associated functions. Research has shown that cellular events, such as signal transduction and gene transcription, require the assembly of proteins into specific macromolecular complexes. Traditional biophysical or biochemical methods have not provided direct access to interactions of protein partners in their natural environments but light microscopic techniques allow us to study molecules under physiological conditions. New imaging technologies, coupled with the development of new genetically encoded fluorescent labels and sensors, and the increasing capability of computer software for image acquisition and analysis, have enabled researchers to conduct more sophisticated studies of the functions and processes of protein molecules, ranging from gene expression to second-messenger cascades and intercellular signaling (DelPozo et al., 2002;Struck et al., 1981;Roessel and Brand, 2002;Ting et al., 2001). One highly sensitive and non-invasive method of protein molecular imaging is Forster (fluorescence) resonance energy transfer (FRET) microscopy. FRET is a distance-dependent physical process, where energy is transferred nonradiatively from an excited molecular fluorophore (donor) to another fluorophore (acceptor) by means of intermolecular longrange dipole-dipole coupling. FRET can accurately measure molecular proximity (1-10 nm), typically when donor and acceptor are positioned within the Forster radius (the distance at which half the excitation energy of the donor is transferred to the acceptor, ~3-6 nm). The efficiency of FRET is dependent on the inverse sixth power of intermolecular separation (Forster, 1965;Lakowicz, 1999;Stryer, 1978), making it a sensitive method for investigating a variety of biological phenomena that produce changes in molecular proximity (Cummings et al., 2002;Day et al 2003;Miyawaki et al., 1999;Wallrabe et al., 2003a). If FRET occurs, donor fluorescence is quenched and acceptor fluorescence is sensitized (increased) (Periasamy and Day, 2005). Co-localization of the donor- and acceptor-labeled probes can be seen within ~0.09 um and molecular associations at close distances can be verified.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL101871-04
Application #
8607064
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
Project End
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
4
Fiscal Year
2013
Total Cost
$189,405
Indirect Cost
$69,806
Name
Case Western Reserve University
Department
Type
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Gaston, Benjamin; May, Walter J; Sullivan, Spencer et al. (2014) Essential role of hemoglobin beta-93-cysteine in posthypoxia facilitation of breathing in conscious mice. J Appl Physiol (1985) 116:1290-9
Davisson, Robin L; Bates, James N; Johnson, Alan Kim et al. (2014) Effects of intracerebroventricular injections of 5-HT on systemic vascular resistances of conscious rats. Microvasc Res 95:116-23
Zaman, Khalequz; Bennett, Deric; Fraser-Butler, Maya et al. (2014) S-Nitrosothiols increases cystic fibrosis transmembrane regulator expression and maturation in the cell surface. Biochem Biophys Res Commun 443:1257-62
Getsy, Paulina M; Davis, Jesse; Coffee, Gregory A et al. (2014) Enhanced non-eupneic breathing following hypoxic, hypercapnic or hypoxic-hypercapnic gas challenges in conscious mice. Respir Physiol Neurobiol 204:147-59
Mendoza, James P; Passafaro, Rachael J; Baby, Santhosh M et al. (2014) Role of nitric oxide-containing factors in the ventilatory and cardiovascular responses elicited by hypoxic challenge in isoflurane-anesthetized rats. J Appl Physiol (1985) 116:1371-81
Palmer, Lisa A; May, Walter J; deRonde, Kimberly et al. (2013) Ventilatory responses during and following exposure to a hypoxic challenge in conscious mice deficient or null in S-nitrosoglutathione reductase. Respir Physiol Neurobiol 185:571-81
Sun, Yuansheng; Rombola, Christina; Jyothikumar, Vinod et al. (2013) Forster resonance energy transfer microscopy and spectroscopy for localizing protein-protein interactions in living cells. Cytometry A 83:780-93
Palmer, Lisa A; May, Walter J; deRonde, Kimberly et al. (2013) Hypoxia-induced ventilatory responses in conscious mice: gender differences in ventilatory roll-off and facilitation. Respir Physiol Neurobiol 185:497-505
Jyothikumar, Vinod; Sun, Yuansheng; Periasamy, Ammasi (2013) Investigation of tryptophan-NADH interactions in live human cells using three-photon fluorescence lifetime imaging and Forster resonance energy transfer microscopy. J Biomed Opt 18:060501
Marozkina, Nadzeya V; Wei, Christina; Yemen, Sean et al. (2012) S-nitrosoglutathione reductase in human lung cancer. Am J Respir Cell Mol Biol 46:63-70

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