The Use of Fluorescent Resonance Energy Transfer to Visualize Nitric Oxide Based Signaling Events in Living Systems Department of Environmental and Occupational Health University of Pittsburgh Graduate School of Public Health Claudette M. St. Croix, PhD
S-Nitrosation and Cell Signaling Broillet Cell Mol Life Sci 55:1036, 1999
Metallothionein Kägi & Vasak, University of Zürich
Cellular Function of Metallothionein Metal Ion Homeostasis: sequestering and transferring Zn, Cu, Cd. Antioxidant: • Elevated oxidative stress • Intercepts O2•-, HO•, •NO, ONOO- • Genetic manipulation studies show that MT can protects cells and tissues against oxidative and nitrosative damage.
Fluorescence Resonance Energy Transfer (FRET) • Non-radiative transfer of excited state energy of the donor to a second light absorbing molecule (acceptor). • Acceptor releases energy through its characteristic fluorescent emission.
Fluorescence Resonance Energy Transfer Detection of NO using metallothionein and two GFP mutants: In the presence of metal, MT folds allowing efficient FRET between ECFP and EYFP fused at either end of MT. In the presence of NO (or a chelating agent) the protein unfolds and FRET is reduced.
Wide-field FRET Widefield FRET using standard Cyan/Yellow fluorescent proteins is difficult because of bleed-through from Cyan emission into yellow FP (25%) While controls such as acceptor photobleaching are critical, methods such as spectral unmixing have proven invaluable. (3 confocal options and one wide field option currently)
Spectral Confocal and FRET This is the Zeiss META design, equivalent though different systems are available from Leica and Olympus
S-nitroso-L-cysteine modifies metallothionein Control L-SNCEE FRET-MT Reporter Cyan Emission Yellow Emission Both St Croix CM. Free Radic Biol Med. 37:785-92, 2004
Baseline SNCEE 5 min SNCEE 10 min TPEN L-SNCEE increases labile Zn in pulmonary endothelial cells
S-nitroso-L-cysteine-EE mediated changes in FRET-MT are reversible St Croix CM. Free Radic Biol Med. 37:785-92, 2004
cGMP Reporter (cygnet-2) Honda et al. PNAS 98:2437, 2001
S-nitroso-L-cysteine activates sGC Cyan Emission Yellow Emission Both cGMP Reporter Control L-SNCEE
Effects of SNCEE on FRET-MT are unaffected by HbO2 St Croix CM. Free Radic Biol Med. 37:785-92, 2004
Tie2-GFP Cy5-Alb Imaging isolated, perfused mouse lung 20X
Expression of FRET-MT in fixed mouse pulmonary endothelium PECAM FRET-MT DAPI Pulmonary gene transfer via tail vein injection of DOTAP:cholesterol liposomes followed by adenovirus containing cDNA for FRET-MT.
Expression of FRET-MT in isolated perfused mouse lung Cy5-Albumin FRET-MT Overlay
Before After Acceptor photo-bleaching confirms that FRET-MT is functional in the perfused lung
DETA NONOate (500 mM) Carbachol (10 mM) Normalized Emission Intensity change in FRET Ratio = 40.7% change in FRET Ratio = 10.7% change in FRET Ratio = 12.2% Emission Wavelength (nm) FRET-MT is regulated by NO in pulmonary endothelium of the intact, perfused mouse lung
NO donors also increase labile zinc in the intact perfused mouse lung A: Zinquin, pretreatment, pseudocolored 2P section in ex vivo perfused murine lung. B: ZnCl2 (+ the zinc ionophore, pyrithione) to perfusate C: attenuation by the zinc chelator, TPEN D: addition of NO donor PAPA nonoate was added to the perfusate (500 M)
Acute hypoxia and pulmonary vasoconstriction von Euler & Liliestrand, 1946
Small vessels of the subpleural vasculature constrict in response to hypoxia
MT -/- mice show no increase in labile zinc in response to hypoxia and have a blunted HPV
Hypoxia Baseline Cyan Emission Yellow Emission Both Hypoxia modifies FRET.MT in cultured endothelial cells FRET.MT Reporter Emission Wavelength (nm) Emission Wavelength (nm)
Hypoxia regulates FRET.MT in the IPL of MT wild-type mice Normalized Emission Intensity
Hpoxia increases labile zinc in the isolated perfused lung of MT wild-type mice
Summary • Hypoxia induces increases in labile zinc in the endothelium of small diameter vessels. • Pharmacologic (e.g. zinc-specific chelator, TPEN) and genetic (targeted ablation of zinc regulatory protein, MT) inhibition of hypoxic mediated elevations in free Zn significantly blunt HPV. • Hypoxia-induced increases in NO synthesis contribute to HPV via formation of S-nitrosothiol in the metal binding center of MT and resultant changes in zinc homeostasis.
Acknowledgements • Center for Biological Imaging • Simon C. Watkins • Glenn Papworth • Surgery • Detcho Stoyanovsky • Roger Y. Tsien (UC-San Diego) • EOH • Bruce R. Pitt • Karanee Leelavanichkul • Zi-Lue Tang • Karla J. Wasserloos • Molly S. Stitt • Xianghong Lui • Pharmacy • Song Li • Annette Wilson