The Use of Fluorescent Resonance Energy Transfer to Visualize Nitric Oxide Based Signaling Events in Living Systems

1
The Use of Fluorescent Resonance Energy Transfer
to Visualize Nitric Oxide Based Signaling Events
in Living Systems
Claudette M. St. Croix, PhD

• Department of Environmental and Occupational
  Health
• University of Pittsburgh Graduate School of
  Public Health

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S-Nitrosation and Cell Signaling
Broillet Cell Mol Life Sci 551036, 1999
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Metallothionein
Kägi Vasak, University of Zürich
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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.

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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.

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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.
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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)
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Spectral Confocal and FRET This is the Zeiss
META design, equivalent though different systems
are available from Leica and Olympus
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S-nitroso-L-cysteine modifies metallothionein
Control L-SNCEE
FRET-MT Reporter
Cyan Emission
Yellow Emission
Both
St Croix CM. Free Radic Biol Med. 37785-92, 2004
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L-SNCEE increases labile Zn in pulmonary
endothelial cells
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S-nitroso-L-cysteine-EE mediated changes in
FRET-MT are reversible
St Croix CM. Free Radic Biol Med. 37785-92, 2004
12
cGMP Reporter (cygnet-2)
Honda et al. PNAS 982437, 2001
13
S-nitroso-L-cysteine activates sGC
Cyan Emission
Yellow Emission
Both
cGMP Reporter
Control
L-SNCEE
14
Effects of SNCEE on FRET-MT are unaffected by
HbO2
St Croix CM. Free Radic Biol Med. 37785-92, 2004
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Imaging isolated, perfused mouse lung
20X
16
(No Transcript)
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Expression of FRET-MT in fixed mouse pulmonary
endothelium
Pulmonary gene transfer via tail vein injection
of DOTAPcholesterol liposomes followed by
adenovirus containing cDNA for FRET-MT.
18
Expression of FRET-MT in isolated perfused mouse
lung
Cy5-Albumin
FRET-MT
Overlay
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Acceptor photo-bleaching confirms that FRET-MT
is functional in the perfused lung
20

FRET-MT is regulated by NO in pulmonary
endothelium of the intact, perfused mouse lung
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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)
22
Pearce, LL Proc Natl Acad Sci 97 477-482, 2000.
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Acute hypoxia and pulmonary vasoconstriction
von Euler Liliestrand, 1946
24
Small vessels of the subpleural vasculature
constrict in response to hypoxia
25
MT -/- mice show no increase in labile zinc in
response to hypoxia and have a blunted HPV
26
Hypoxia modifies FRET.MT in cultured endothelial
cells
FRET.MT Reporter
Emission Wavelength (nm)
Emission Wavelength (nm)
27
Hypoxia regulates FRET.MT in the IPL of MT
wild-type mice
Normalized Emission Intensity
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Hypoxia-induced increases in zinc are dependent
on NO
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Hpoxia increases labile zinc in the isolated
perfused lung of MT wild-type mice
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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.

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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