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New Techniques to Study Intracellular Receptors in Living Cells: Insights Into RIG-I-Like Receptor Signaling

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Protein Reviews – Purinergic Receptors

Part of the book series: Advances in Experimental Medicine and Biology ((PROTRE,volume 1111))

Abstract

This review discusses new developments in Förster resonance energy transfer (FRET) microscopy and its application to cellular receptors. The method is based on the kinetic theory of FRET, which can be used to predict FRET not only in dimers, but also higher order oligomers of donor and acceptor fluorophores. Models based on such FRET predictions can be fit to observed FRET efficiency histograms (also called FRET spectrograms) and used to estimate intracellular binding constants, free energy values, and stoichiometries. These “FRET spectrometry” methods have been used to analyze oligomers formed by various receptors in cell signaling pathways, but until recently such studies were limited to receptors residing on the cell surface. To study complexes residing inside the cell, a technique called Quantitative Micro-Spectroscopic Imaging (Q-MSI) was developed. Q-MSI combines determination of quaternary structure from pixel-level apparent FRET spectrograms with the determination of both donor and acceptor concentrations at the organelle level. This is done by resolving and analyzing the spectrum of a third fluorescent marker, which does not participate in FRET. Q-MSI was first used to study the interaction of a class of cytoplasmic receptors that bind viral RNA and signal an antiviral response via complexes formed mainly on mitochondrial membranes. Q-MSI revealed previously unknown RNA mitochondrial receptor orientations, and the interaction between the viral RNA receptor called LGP2 with the RNA helicase encoded by the hepatitis virus. The biological importance of these new observations is discussed.

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Abbreviations

CARD:

Capsase activation and recruitment domain

CFP:

Cyan fluorescent protein

FP:

Fluorescent protein

FRET:

Förster resonance energy transfer

FSI:

Fully quantitative spectral imaging

GFP:

Green fluorescent protein

HCV:

Hepatitis C virus

LGP2:

Laboratory of genetics and physiology-2

MAVS:

Mitochondrial antiviral signaling protein

MDA5:

Melanoma differentiated antigen-5

NS3:

Non-structural protein 3

NS4A:

Non-structural protein 4A

PAMP:

Pathogen associated molecular pattern

Poly(I:C):

Polyinosinic: polycytidylic acid

Q-MSI:

Quantitative micro-spectroscopic imaging

RIG-I:

Retinoic inducible gene-I

RLR:

RIG-I like receptor

ROI:

Region of interest

YFP:

Yellow fluorescent protein

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Acknowledgements

Funding for this research was provided by the National Institutes of Health (Grant No. RO1AI088001 awarded to DNF), UWM Research Growth Innitiative (Grant No. 101X333 awarded to DNF and VR) and the National Science Foundation, Major Research Instrumentation Program (Grants No. PHY-1126386 and PHY-1626450 awarded to V.R.).

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI, USA

    M. J. Corby

  2. Departments of Physics and Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI, USA

    Valerica Raicu

  3. Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI, USA

    David N. Frick

Authors
  1. M. J. Corby
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  2. Valerica Raicu
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  3. David N. Frick
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Corresponding authors

Correspondence to Valerica Raicu or David N. Frick .

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Editors and Affiliations

  1. Biochem and Mol Biol, Baylor College of Medicine, Houston, TX, USA

    M. Zouhair Atassi

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Corby, M.J., Raicu, V., Frick, D.N. (2018). New Techniques to Study Intracellular Receptors in Living Cells: Insights Into RIG-I-Like Receptor Signaling. In: Atassi, M. (eds) Protein Reviews – Purinergic Receptors. Advances in Experimental Medicine and Biology(), vol 1111. Springer, Cham. https://doi.org/10.1007/5584_2018_297

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