Adv Exp Med Biol - Protein Reviews https://doi.org/10.1007/5584_2018_297 # Springer Nature Switzerland AG 2018 New Techniques to Study Intracellular Receptors in Living Cells: Insights Into RIG-I-Like Receptor Signaling M.J. Corby, Valerica Raicu, and David N. Frick Abstract determination of quaternary structure from This review discusses new developments in pixel-level apparent FRET spectrograms with Förster resonance energy transfer (FRET) the determination of both donor and acceptor microscopy and its application to cellular concentrations at the organelle level. This is receptors. The method is based on the kinetic done by resolving and analyzing the spectrum theory of FRET, which can be used to predict of a third fluorescent marker, which does not FRET not only in dimers, but also higher order participate in FRET. Q-MSI was first used to oligomers of donor and acceptor fluorophores. study the interaction of a class of cytoplasmic Models based on such FRET predictions can be receptors that bind viral RNA and signal an fit to observed FRET efficiency histograms antiviral response via complexes formed mainly (also called FRET spectrograms) and used to on mitochondrial membranes. Q-MSI revealed estimate intracellular binding constants, free previously unknown RNA mitochondrial energy values, and stoichiometries. These receptor orientations, and the interaction “FRET spectrometry” methods have been between the viral RNA receptor called LGP2 used to analyze oligomers formed by various with the RNA helicase encoded by the hepatitis receptors in cell signaling pathways, but until virus. The biological importance of these new recently such studies were limited to receptors observations is discussed. residing on the cell surface. To study complexes residing inside the cell, a technique called Keywords Quantitative Micro-Spectroscopic Imaging Antiviral response · ATPase · FRET · Hepatitis (Q-MSI) was developed. Q-MSI combines C virus · Innate immunity · LGP2 · MDA5 · RIG-I · RNA helicase M. J. Corby Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI, USA V. Raicu (*) Abbreviations Departments of Physics and Biological Sciences, CARD Capsase activation and recruitment University of Wisconsin-Milwaukee, Milwaukee, WI, domain USA fl e-mail: [email protected] CFP Cyan uorescent protein FP Fluorescent protein D. N. Frick (*) Department of Chemistry and Biochemistry, University of FRET Förster resonance energy transfer Wisconsin-Milwaukee, Milwaukee, WI, USA FSI Fully quantitative spectral imaging e-mail: [email protected] M. J. Corby et al. GFP Green fluorescent protein and Schmidt 2017). This review discusses such HCV Hepatitis C virus FRET techniques, and how they have been used to LGP2 Laboratory of genetics and physiology-2 analyze oligomers formed by various receptors in MAVS Mitochondrial antiviral signaling protein cell signaling pathways. Since much of this work MDA5 Melanoma differentiated antigen-5 with cell surface receptors was recently reviewed NS3 Non-structural protein 3 elsewhere (Raicu and Singh 2013; Raicu and NS4A Non-structural protein 4A Schmidt 2017), the focus here is mainly on the PAMP Pathogen associated molecular pattern latest FRET methods designed to analyze Poly Polyinosinic: polycytidylic acid receptors located in the cytoplasm or intracellular (I:C) organelles, in particular. The receptors discussed Q-MSI Quantitative micro-spectroscopic imaging here are called RIG-I like receptors (RLRs). RIG-I Retinoic inducible gene-I RLRs bind cytoplasmic nucleic acids known RLR RIG-I like receptor to be pathogen associated molecular patterns ROI Region of interest (PAMPs). Once activated, RLRs change confor- YFP Yellow fluorescent protein mation and signal through downstream enzymes to initiate transcription of interferons and other antiviral proteins (Fig. 1). RLRs locate their cyto- plasmic ligands using motor domains resembling those found in helicases that separate the DNA 1 Introduction double helix by disrupting Watson-Crick base pairs in an ATP-fueled reaction. The RLR Förster resonance energy transfer (FRET) helicases do not separate complementary strands, (Forster 1946) has been used in microscopy for but instead scan cytoplasmic RNA to identify many years to study molecular interactions. “non-self” motifs indicative of a viral infection. FRET assays monitor the transfer of energy The FRET methods discussed rely on the anal- from a “donor” fluorophore to an “acceptor” ysis of image stacks acquired at a series of emis- fluorophore, which need to be within 10 nm sion wavelengths such that a spectrum is resolved (100 Å). Because FRET efficiency depends on at each pixel (Biener et al. 2013). These methods, the sixth power of the distance between the termed “optical micro-spectroscopy,” can be donor and acceptor (Stryer and Haugland 1967), performed using commercially available two- FRET microscopy has been used to estimate the photon (Biener et al. 2013) and possibly confocal relative distances between two macromolecules, microscopes with spectral resolution (Zimmerman typically by fusing each to a different fluorescent et al. 2003). The main focus here involves the protein (FP). Introduction of the kinetic theory of recent development of a technique called Quantita- FRET (Raicu 2007) allowed one to predict FRET tive Micro-Spectroscopic Imaging (Q-MSI) efficiencies in situations where more than one (Stoneman et al. 2017; Mishra et al. 2016; donor or acceptor participates in FRET and has Corby et al. 2017). Q-MSI combines determina- expanded the amount of information that can be tion of quaternary structure from pixel-level obtained using FRET-based microscopy apparent FRET efficiency histograms (also (Patowary et al. 2015; King et al. 2017). For known as FRET spectrograms (Raicu and Singh example, donor and acceptor concentrations, and 2013)) with the determination of both donor and pixel-level FRET efficiencies can be determined acceptor concentrations at the organelle level in various subcellular locations (Raicu et al. (King et al. 2016). Q-MSI enables the ability to 2009). These data can be used with the Law of visualize the sub-cellular locations of both donors Mass Action to estimate intracellular binding and acceptors by resolving and analyzing the constants, free energy values, and stoichiometries, spectrum of a third fluorescent marker, which and provide clues to the quaternary structure of does not participate in FRET. oligomeric complexes (King et al. 2016; Raicu New Techniques to Study Intracellular Receptors in Living Cells: Insights... RIG-I MDA5 ± MAVS LGP2 TRAF2/6 NEMO TRAF3 NEMO RIP1 IKK TANK TBK1 IKK IRF3 IKK IRF7 NF- B Interferons IRSE & Cytokines Fig. 1 Oligomer formation in RLR signaling. Various leads to a conformational change exposing CARDs (pur- RNA viruses (red, blue) activate antiviral genes after being ple) that are ubiquitinated (green) and seed oligomers detected through the RLRs (RIG-I, MDA5, & LGP2) formed by RLRs and a mitochondrial antiviral signaling which all converge on MAVS (grey). Ligand binding protein (MAVS) emission spectrum and the acceptor absorption 2 Overview of FRET spectrum is optimized while minimizing any direct excitation by the acceptor at the donor FRET between FPs fused to cellular proteins has excitation wavelength. In addition, FPs must be been measured using microscopes for over fused to target proteins so that they are not dia- 20 years, and numerous comprehensive reviews metrically opposed in a complex, and so that they on the topic are available (Jares-Erijman and do not block key motifs needed for protein- Jovin 2003; Piston and Kremers 2007; Shaner protein interactions. FRET can be detected using et al. 2005; Padilla-Parra and Tramier 2012; various methods including filter sets designed to Bajar et al. 2016). Briefly, successful FRET detect donor and acceptor fluorescence at one or experiments depend on the selection of an appro- two excitation wavelengths (Xia and Liu 2001; priate donor/acceptor FP pair. For example if Hoppe et al. 2002), acceptor photobleaching Aequorea victoria GFP derivatives are used, the (Tramier et al. 2006), fluorescence lifetime imag- donor FP could be a green (GFP, or GFP2) or ing (Lakowicz et al. 1992), changes in florescence cyan (Cerulean or Sapphire) variant, while the polarization (Mattheyses et al. 2004), or by spec- acceptor could be a yellow variant, such as YFP, tral imaging (Zimmermann et al. 2002). The Citrine, or Venus. The donor should have a high strengths and weaknesses of each of these quantum yield, while the excitation spectrum of techniques have been rigorously discussed else- the acceptor FP should have the highest spectral where (Jares-Erijman and Jovin 2003; Piston and overlap possible with the donor emission spec- Kremers 2007; Shaner et al. 2005; Padilla-Parra trum, to ensure that it is able to receive the largest and Tramier 2012; Leavesley et al. 2013; Bajar number of excitations from the donor. In most et al. 2016; Raicu and Singh 2013). The methods cases, it is best if the overlap between the donor M. J. Corby et al. below are based exclusively on spectral FRET excitation scans to determine the FRET effi- imaging microscopy (Chen et al. 2007; Raicu ciency, the length of time (~60 s) needed to col- et al. 2005, 2009). lect those scans leaves times for molecular diffusion between scans to scramble the molecu- lar makeup of the sample at the point of excitation and hence the FRET efficiency may only be cal- 2.1 Optical Micro-spectroscopy culated as an average over many pixels (i.e.,