The F-techniques: advances in receptor protein studies

doi:10.1016/j.tem.2008.02.004

Trends in Endocrinology & Metabolism

Volume 19, Issue 5, July 2008, Pages 181-190

https://doi.org/10.1016/j.tem.2008.02.004 Get rights and content

Recent developments in advanced microscopy techniques, the so-called F-techniques, including Förster resonance energy transfer, fluorescence correlation spectroscopy and fluorescence lifetime imaging, have led to a wide range of novel applications in biology. The F-techniques provide quantitative information on biomolecules and their interactions and give high spatial and temporal resolution. In particular, their application to receptor protein studies has led to new insights into receptor localization, oligomerization, activation and function in vivo. This review focuses on the application of the F-techniques to the study of receptor molecules and mechanisms in the last three years and provides information on new modalities that will further improve their applicability and widen the range of biological questions that can be addressed.

Introduction

The maintenance and survival of multicellular organisms require an efficient system of cell–cell communication to coordinate the wide range of physiological processes that occur within an organism. This communication is performed by signaling pathways that are initiated by ligands binding to their target membrane receptors. The receptor–ligand complex then transduces the message to the cell interior. Because physiological and molecular functions measured in vitro and in vivo can show significant differences [1], it is necessary to measure receptor function in vivo over extended periods with high temporal and spatial resolution. Fluorescence techniques provide several advantages compared with other biophysical and biochemical methods for measuring receptor function. First, fluorescence intensity is linearly dependent on the number of fluorophores in a sample, providing a basis for quantitative measurements. Second, fluorescence measurements possess a very high sensitivity and thus can be performed on single molecules, providing the opportunity to observe biological mechanisms on a molecular level. Third, fluorescence is a process characterized by a range of different parameters, which can be measured independently or in combination (e.g. intensity, wavelength, fluorophore lifetime, polarization), providing information not only on the mere presence of a fluorophore but also on its orientation and its immediate environment. And finally, these techniques can be used in live cells. Here, we review the progress made over the last three years in fluorescence techniques for the study of receptor proteins. We concentrate on advanced fluorescence techniques, termed F-techniques, including Förster resonance energy transfer (FRET), fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS, respectively) and fluorescence lifetime imaging (FLIM) and their application to the observation of receptor protein localization, oligomerization, activation and function in vivo.

Section snippets

Förster or fluorescence resonance energy transfer (FRET)

FRET, the molecular process of energy transfer between two molecules, was first described by Förster in 1948 [2]. This technique can be used to measure the proximity of two fluorescently labeled molecules at a distance of less than 10 nm, by measuring the FRET efficiency, which is strongly dependent on the distance between the molecules (Box 1). Since the usefulness of FRET as a molecular ruler was shown in 1967 by Stryer and Haugland [3], FRET has developed into one of the preferred methods for

Fluorescence lifetime imaging microscopy (FLIM)

FLIM was first used in cells to measure the spatial distribution of fluorescence lifetimes [44] (Box 2). Because the lifetime of a fluorophore depends on its local environment, any parameter that has an influence on the lifetime can be mapped with optical resolution. Polarity, pH, ion concentrations or energy transfer between fluorophores can be measured, depending on the fluorophores used and their sensitivity to particular parameters. For a general review on FLIM instrumentation and usages,

Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS)

FCS and FCCS are single-molecule-sensitive techniques that record and evaluate the fluorescence signal from a small observation volume (Box 3). The review by Krichevsky and Bonnet discusses FCS [59], and FCCS has been reviewed in Refs 60, 61. The power of FCS lies in its access to processes on the single molecule level, while at the same time statistically evaluating many single events via correlation functions. The most common parameters extracted from FCS are the diffusion coefficient or flow

Fluorophores

Fluorescence labeling of biomolecules in vivo is difficult and requires extensive tests on possible influences of the labeling on protein function. Using several labeling approaches in parallel is therefore often a very time consuming, and sometimes unrealistic, option. Therefore, in recent years, researchers have adapted the F-techniques to the most common fluorescent tags even though these tags might not always be the best choice in terms of signal-to-noise ratio. One of the most common

Conclusions

It is clear that the F-techniques have been able to resolve important questions regarding the role of protein dynamics in biology. Table 1 summarizes the characteristics as well as the applications of the F-techniques in recent years. Instruments are commercially available to measure FRET, FLIM and FCS, but new developments occur at an unprecedented rate, and measurements are invariably facilitated by customized instruments. The application of the F-techniques to three-dimensional systems, such

Acknowledgement

This work was funded by the Singapore Bioimaging Consortium (SBIC-003/2005). We thank Xianke Shi and Sudhaharan Thankiah for cell images.

References (91)

D.B. VanBeek
Fretting about FRET: Correlation between κ and R
Biophys. J.
(2007)
D. Sahoo
Scavenger receptor class B, type I (SR-BI) homo-dimerizes via its C-terminal region: Fluorescence resonance energy transfer analysis
Biochim. Biophys. Acta
(2007)
K. Herrick-Davis
Serotonin 5-HT2C receptor homodimer biogenesis in the endoplasmic reticulum: Real-time visualization with confocal fluorescence resonance energy transfer
J. Biol. Chem.
(2006)
S. Tenhumberg
gp130 dimerization in the absence of ligand: Preformed cytokine receptor complexes
Biochem. Biophys. Res. Commun.
(2006)
T.C. Cheung et al.
Dimerizations of the wallaby gonadotropin-releasing hormone receptor and its splice variants
Gen. Comp. Endocrinol.
(2005)
K. Herrick-Davis
Inhibition of serotonin 5-hydroxytryptamine2C receptor function through heterodimerization: Receptor dimers bind two molecules of ligand and one G-protein
J. Biol. Chem.
(2005)
M. Du
Conformational changes in the ligand-binding domain of a functional ionotropic glutamate receptor
J. Biol. Chem.
(2005)
J. Nakanishi
FRET-based monitoring of conformational change of the beta(2) adrenergic receptor in living cells
Biochem. Biophys. Res. Commun.
(2006)
S. Granier
Structure and conformational changes in the C-terminal domain of the beta(2)-adrenoceptor: Insights from fluorescence resonance energy transfer studies
J. Biol. Chem.
(2007)
H.A. Hostetler
Peroxisome proliferator-activated receptor alpha interacts with high affinity and is conformationally responsive to endogenous ligands
J. Biol. Chem.
(2005)

P. Hernanz-Falcon

Identification of amino acid residues crucial for chemokine receptor dimerization

Nat. Immunol.

(2004)

G. Ramanoudjame

Allosteric mechanism in AMPA receptors: A FRET-based investigation of conformational changes

Proc. Natl Acad. Sci. USA

(2006)

Cited by (27)

Automated selection of regions of interest for intensity-based FRET analysis of transferrin endocytic trafficking in normal vs. cancer cells
2014, Methods
Citation Excerpt :
Therefore, the development of novel quantitative imaging techniques is crucial to increase our understanding of the regulation of the intracellular endocytic trafficking of membrane receptors in both normal and cancer cells. Recently, Förster resonance energy transfer (FRET)-based imaging approaches have been developed to assay receptor dimerization/oligomerization as well as receptor–ligand interactions during endocytic trafficking pathways [13–16]. In particular, a quantitative FRET assay has been used to follow the endocytic/recycling trafficking of transferrin receptor (TFR) and its respective ligand, iron-bound transferrin (Tfn), at nanometer range resolution [17–22].
The overexpression of certain membrane-bound receptors is a hallmark of cancer progression and it has been suggested to affect the organization, activation, recycling and down-regulation of receptor–ligand complexes in human cancer cells. Thus, comparing receptor trafficking pathways in normal vs. cancer cells requires the ability to image cells expressing dramatically different receptor expression levels. Here, we have presented a significant technical advance to the analysis and processing of images collected using intensity based Förster resonance energy transfer (FRET) confocal microscopy. An automated Image J macro was developed to select region of interests (ROI) based on intensity and statistical-based thresholds within cellular images with reduced FRET signal. Furthermore, SSMD (strictly standardized mean differences), a statistical signal-to-noise ratio (SNR) evaluation parameter, was used to validate the quality of FRET analysis, in particular of ROI database selection. The Image J ROI selection macro together with SSMD as an evaluation parameter of SNR levels, were used to investigate the endocytic recycling of Tfn–TFR complexes at nanometer range resolution in human normal vs. breast cancer cells expressing significantly different levels of endogenous TFR. Here, the FRET-based assay demonstrates that Tfn–TFR complexes in normal epithelial vs. breast cancer cells show a significantly different E% behavior during their endocytic recycling pathway. Since E% is a relative measure of distance, we propose that these changes in E% levels represent conformational changes in Tfn–TFR complexes during endocytic pathway. Thus, our results indicate that Tfn–TFR complexes undergo different conformational changes in normal vs. cancer cells, indicating that the organization of Tfn–TFR complexes at the nanometer range is significantly altered during the endocytic recycling pathway in cancer cells. In summary, improvements in the automated selection of FRET ROI datasets allowed us to detect significant changes in E% with potential biological significance in human normal vs. cancer cells.
Insights into subunit interactions within the insect olfactory receptor complex using FRET
2013, Insect Biochemistry and Molecular Biology
Citation Excerpt :
Typically the fluorophores are modified versions of the Green Fluorescent Protein, such as Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP), which are fused to the protein of interest. For integral membrane proteins, resonance energy transfer methods (e.g. FRET and BRET) have been used to detect homomeric and heteromeric interactions among receptor subunits, with several examples from GPCRs (Liu et al., 2008; Mercier et al., 2002; Pfleger and Eidne, 2005). We use intermolecular FRET by acceptor photobleaching to confirm homomeric and heteromeric interactions between Orco and the ligand-binding OR, Or22a.
Insect olfactory receptors (ORs) are a novel family of ligand-gated cation channels that can respond to volatile organic compounds at low concentrations. They are involved in the detection of odorants associated with mate recognition, food localisation and predator avoidance. These receptors form a complex that is currently thought to contain at least two subunit members: the non-canonical Orco ion channel subunit and a ligand-binding receptor subunit. The integral membrane proteins SNMP1 and 2 are also associated with olfactory function, with SNMP1 required for cis-vaccinyl acetate reception in Drosophila melanogaster. In order to investigate protein–protein interactions among these membrane proteins we measured intermolecular Förster/Fluorescence Resonance Energy Transfer (FRET) in live insect cells by acceptor photobleaching. Fusion proteins containing Cyan Fluorescent Protein or Yellow Fluorescent Protein were produced using baculovirus-mediated expression in High Five™ cells. The majority of the recombinant products were of the expected size for the fusion proteins and located within intracellular membranes. We were able to show FRET efficiencies providing evidence for homomeric and heteromeric interactions of the ligand-binding OR, Or22a, and Orco (Or22a–Or22a, Or22a–Orco, Orco–Orco). There was no evidence for an interaction between SNMP1 and Orco or between SNMP2 and Orco or Or22a. However, fusion proteins of SNMP1 and Or22a did show an interaction by FRET, suggesting SNMP1 may interact with at least some insect olfactory receptor complexes. In summary, this study supports previously observed homomeric and heteromeric interactions between Orco and the ligand-binding OR, Or22a, and identifies a novel interaction between Or22a and SNMP1.
Single cell analysis of ligand binding and complex formation of interleukin-4 receptor subunits
2011, Biophysical Journal
Citation Excerpt :
There is still medical need for improvement in understanding and manipulating cytokine and growth factor signaling (1). Fostered by the continuous expansion and development of new microscopic fluorescence-based methods in the last decade, cell biologists now have access to a broad spectrum of powerful tools to analyze molecular mechanisms at the single cell level (2–4). Within the various approaches to investigate protein-protein interactions in live cells, fluorescence correlation spectroscopy (FCS) is distinguished by providing dynamic information over a broad range of timescales and single-molecule sensitivity (5).
Interleukin-4 (IL-4) is an important class I cytokine involved in adaptive immunity. IL-4 binds with high affinity to the single-pass transmembrane receptor IL-4Rα. Subsequently, IL-4Rα/IL-4 is believed to engage a second receptor chain, either IL-2Rγ or IL-13Rα1, to form type I or II receptor complexes, respectively. This ternary complex formation then triggers downstream signaling via intracellular Janus kinases bound to the cytoplasmic receptor tails. Here, we study the successive steps of complex formation at the single cell level with confocal fluorescence imaging and correlation spectroscopy. We characterize binding and signaling of fluorescently labeled IL-4 by flow cytometry of IL-4-dependent BaF3 cells. The affinity to ectopically expressed IL-4Rα was then measured by single-color fluorescence correlation spectroscopy in adherent HEK293T cells that express the components of the type II IL-4R but not type I. Finally, IL-4-induced complex formation was tested by dual-color fluorescence cross-correlation spectroscopy. The data provide evidence for codiffusion of IL-4-A647 bound IL-4Rα and the type II subunit IL-13Rα1 fused to enhanced green fluorescent protein, whereas type I complexes containing IL-2Rγ and JAK3 were not detected at the cell surface. This behavior may reflect hitherto undefined differences in the mode of receptor activation between type I (lymphoid) and type II (epithelial) receptor expressing cells.
Spotting the right location- imaging approaches to resolve the intracellular localization of invasive pathogens
2011, Biochimica et Biophysica Acta - General Subjects
A common strategy of microbial pathogens is to invade host cells during infection. The invading microbes explore different intracellular compartments to find their preferred niche.
Imaging has been instrumental to unravel paradigms of pathogen entry, to identify their exact intracellular location, and to understand the underlying mechanisms for the formation of pathogen-containing niches. Here, we provide an overview of imaging techniques that have been applied to monitor the intracellular lifestyle of pathogens, focusing mainly on bacteria that either remain in vacuolar-bound compartments or rupture the endocytic vacuole to escape into the host's cellular cytoplasm.
We will depict common molecular and cellular paradigms that are preferentially exploited by pathogens. A combination of electron microscopy, fluorescence microscopy, and time-lapse microscopy has been the driving force to reveal underlying cell biological processes. Furthermore, the development of highly sensitive and specific fluorescent sensor molecules has allowed for the identification of functional aspects of niche formation by intracellular pathogens.
Currently, we are beginning to understand the sophistication of the invasion strategies used by bacterial pathogens during the infection process- innovative imaging has been a key ingredient for this.
This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.
Developing in vivo biophysics by fishing for single molecules
2010, Developmental Biology
Single-molecule techniques (SMT) provide the possibility to quantitatively analyze the action of single molecules. SMTs can resolve the distribution of states of an ensemble of molecules, collecting information that is otherwise not accessible by typical ensemble techniques. Until now, the application of SMTs in developmental biology was limited. Several recent studies illustrate the possibility to investigate the behavior of single biological molecules in invertebrates such as Caenorhabditis elegans and transparent embryos of model teleosts. These studies have paved the way for the application of fluorescence-based SMTs, e.g. fluorescence correlation spectroscopy, fluorescent energy transfer, or single particle tracking, in developmental biology. This review aims to define SMTs applicable in developmental biology, and discuss properties of an ideal animal model.
Combinatorial microscopy for the study of protein-membrane interactions in supported lipid bilayers: Order parameter measurements by combined polarized TIRFM/AFM
2009, Journal of Structural Biology
Understanding the mechanisms of peptide-induced membrane disorder is critical to the design of novel antimicrobial and cell-penetrating peptides. One means of quantifying local structure and order/disorder is through the orientational order parameter, typically obtained using various spectroscopic approaches. We report here on the use of an image-based means of tracking the order parameter in supported lipid bilayers during peptide-induced disordering. By coupling polarized total internal reflection fluorescence microscopy with in situ atomic force microscopy, it is now possible to track changes in order parameter associated with peptide binding and insertion, as well as lipid headgroup and acyl chain reordering, while simultaneously resolving molecular-scale topographical changes. Interactions between the model antimicrobial peptide, indolicidin, and its fluorescent analog, TAMRA-indolicidin, with model eukaryotic (DOPC:DSPC:cholesterol) and prokaryotic (DOPE/DOPG) membranes were tracked using the fluorescent lipid reporters, DiI-C₂₀ and BODIPY-PC. Changes in the order parameter upon membrane binding and insertion provided insights into the orientation of the peptide and the role of membrane chemistry and composition on insertion dynamics and membrane restructuring.

View all citing articles on Scopus

View full text

Trends in Endocrinology & Metabolism

ReviewThe F-techniques: advances in receptor protein studies

Introduction

Section snippets

Förster or fluorescence resonance energy transfer (FRET)

Fluorescence lifetime imaging microscopy (FLIM)

Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS)

Fluorophores

Conclusions

Acknowledgement

Biophys. J.

Biochim. Biophys. Acta

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Gen. Comp. Endocrinol.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Trends Endocrinol. Metab.

Biophys. J.

Vision Res.

Biophys. J.

Cell Calcium

Arch. Biochem. Biophys.

Biophys. J.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biophys. J.

Blood

Biophys. J.

Biophys. J.

J. Biol. Chem.

Exp. Mol. Pathol.

Biophys. J.

Biophys. J.

Biophys. J.

Biophys. J.

Navigating chemical space for biology and medicine

Nature

Intermolecular energy migration and fluorescence

Ann. Phys. (Leipzig)

Energy transfer: a spectroscopic ruler

Proc. Natl Acad. Sci. USA

Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments

Nat. Methods

Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

Nat. Struct. Mol. Biol.

Fluorescence resonance energy transfer (FRET) microscopy in living cells as a novel tool for the study of cytokine action

J. Dairy Res.

Heterotrimeric G proteins precouple with G protein-coupled receptors in living cells

Proc. Natl Acad. Sci. USA

Dimerization of corticotropin-releasing factor receptor type 1 is not coupled to ligand binding

J. Recept. Signal Transduct. Res.

The specificity and molecular basis of alpha(1)-adrenoceptor and CXCR chemokine receptor dimerization

J. Mol. Neurosci.

FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells

Proc. Natl Acad. Sci. USA

Dimerization of the cytokine receptors gp130 and LIFR analysed in single cells

J. Cell Sci.

Quantitative FRET imaging of leptin receptor oligomerization kinetics in single cells

Biol. Cell

Cutting edge: Evidence for ligand-independent multimerization of the IL-17 receptor

J. Immunol.

Identification of amino acid residues crucial for chemokine receptor dimerization

Nat. Immunol.

Allosteric mechanism in AMPA receptors: A FRET-based investigation of conformational changes

Proc. Natl Acad. Sci. USA

Review
The F-techniques: advances in receptor protein studies