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The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M

The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M 1 muscarinic acetylcholine receptor was replaced with a C-terminally monomeric enhanced green fluorescent protein (mEGFP)–linked variant. Fluorescence imaging of brain slices demonstrated appropriate regional distribution, and using both anti-M 1 and anti–green fluorescent protein antisera the expressed transgene was detected in both cortex and hippocampus only as the full-length polypeptide. M 1 -mEGFP was expressed at levels equal to the M 1 receptor in wild-type mice and was expressed throughout cell bodies and projections in cultured neurons from these animals. Signaling and behavioral studies demonstrated M 1 -mEGFP was fully active. Application of fluorescence intensity fluctuation spectrometry to regions of interest within M 1 -mEGFP–expressing neurons quantified local levels of expression and showed the receptor was present as a mixture of monomers, dimers, and higher-order oligomeric complexes. Treatment with both an agonist and an antagonist ligand promoted monomerization of the M 1 -mEGFP receptor. The quaternary organization of a class A G protein-coupled receptor in situ was directly quantified in neurons in this study, which answers the much-debated question of the extent and potential ligand-induced regulation of basal quaternary organization of such a receptor in native tissue when present at endogenous expression levels.

Background: The growing evidence that G protein-coupled receptors (GPCRs) not only form oligomers but that the oligomers also may modulate the receptor function provides a promising avenue in the area of drug design. Highly selective drugs targeting distinct oligomeric sub-states offer the potential to increase efficacy while reducing side effects. In this regard, determining the various oligomeric configurations and geometric sub-states of a membrane receptor is of utmost importance. Methods: In this report, we have reviewed two techniques that have proven to be valuable in monitoring the quaternary structure of proteins in vivo: Fӧrster resonance energy transfer (FRET) spectrometry and fluorescence intensity fluctuation (FIF) spectrometry. In FRET spectrometry, distributions of pixel-level FRET efficiency are analyzed using theoretical models of various quaternary structures to determine the geometry and stoichiometry of protein oligomers. In FIF spectrometry, spatial fluctuations of fluorescent molecule intensities are analyzed to reveal quantitative information on the size and stability of protein oligomers. Results: We demonstrate the application of these techniques to a number of different fluorescence-based studies of cells expressing fluorescently labeled membrane receptors, both in the presence and absence of various ligands. The results show the effectiveness of using FRET spectrometry to determine detailed information regarding the quaternary structure receptors form, as well as FIF and FRET for determining the relative abundance of different-sized oligomers when an equilibrium forms between such structures. Conclusion: FRET and FIF spectrometry are valuable techniques for characterizing membrane receptor oligomers, which are of great benefit to structure‐based drug design.

While the notion that G protein-coupled receptors (GPCRs) associate into homo- and hetero-oligomers has gained more recognition in recent years, a lack of consensus remains among researchers regarding the functional relevance of GPCR oligomerization. A technique, Förster resonance energy transfer (FRET) spectrometry, allows for the determination of the oligomeric (or quaternary) structure of proteins in living cells via analysis of efficiency distributions of energy transferred from optically excited fluorescent tags acting as donors of energy to fluorescent tags acting as acceptors of energy and residing within the same oligomer. In this study, we significantly improved the resolution of FRET spectrometry to detect subtle differences in quaternary structures of GPCR oligomers within living cells. We then used this approach to study the conformational substates of oligomers of the sterile 2 α-factor receptor (Ste2), a class D GPCR found in the yeast

Fluorescence fluctuation spectroscopy (FFS) encompasses a bevy of techniques that involve analyzing fluorescence intensity fluctuations occurring due to fluorescently labeled molecules diffusing in and out of a microscope's focal region. Statistical analysis of these fluctuations may reveal the oligomerization (i.e., association) state of said molecules. We have recently developed a new FFS-based method, termed Two-Dimensional Fluorescence Intensity Fluctuation (2D FIF) spectrometry, which provides quantitative information on the size and stability of protein oligomers as a function of receptor concentration. This article describes protocols for employing FIF spectrometry to quantify the oligomerization of a membrane protein of interest, with specific instructions regarding cell preparation, image acquisition, and analysis of images given in detail. Application of the FIF Spectrometry Suite, a software package designed for applying FIF analysis on fluorescence images, is emphasized in the protocol. Also discussed in detail is the identification, removal, and/or analysis of inhomogeneous regions of the membrane that appear as bright spots. The 2D FIF approach is particularly suited to assess the effects of agonists and antagonists on the oligomeric size of membrane receptors of interest. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of live cells expressing protein constructs Basic Protocol 2: Image acquisition and noise correction Basic Protocol 3: Drawing and segmenting regions of interest Basic Protocol 4: Calculating the molecular brightness and concentration of individual image segments Basic Protocol 5: Combining data subsets using a manual procedure (Optional) Alternate Protocol 1: Combining data subsets using the advanced FIF spectrometry suite (Optional; alternative to Basic Protocol 5) Basic Protocol 6: Performing meta-analysis of brightness spectrograms Alternate Protocol 2: Performing meta-analysis of brightness spectrograms (alternative to Basic Protocol 6) Basic Protocol 7: Spot extraction and analysis using a manual procedure or by writing a program (Optional) Alternate Protocol 3: Automated spot extraction and analysis (Optional; alternative to Protocol 7) Support Protocol: Monomeric brightness determination.

Here, we introduce fluorescence intensity fluctuation spectrometry for determining the identity, abundance and stability of protein oligomers. This approach was tested on monomers and oligomers of known sizes and was used to uncover the oligomeric states of the epidermal growth factor receptor and the secretin receptor in the presence and absence of their agonist ligands. This method is fast and is scalable for high-throughput screening of drugs targeting protein–protein interactions.

Fluorescence micrographs of the plasma membrane of cells expressing fluorescently labeled G protein-coupled receptors (GPCRs) often exhibit small clusters of pixels (or puncta) with intensities that are higher than those of the surrounding pixels. Although studies of GPCR interactions in uniform membrane areas abound, understanding the details of the GPCR interactions within such puncta as well as the nature of the membrane formations underlying the puncta is hampered by the lack of adequate experimental techniques. Here, we introduce an enhancement of a recently developed method termed fluorescence intensity fluctuation spectrometry, which permits analysis of protein-protein interactions within the puncta in live cell membranes. We applied the novel fluorescence intensity fluctuation data analysis protocol to previously published data from cells expressing human secretin receptors and determined that the oligomer size increases with receptor concentration and duration of treatment with cognate ligand, not only within uniform regions of the membrane (in agreement with previous publications) but also within the puncta. In addition, we found that the number density and fractional area of the puncta increased after treatment with ligand. This method could be applied for probing the evolution in the time of the chain of events that begins with ligand binding and continues with coated pits formation and receptor internalization for other GPCRs and, indeed, other membrane receptors in living cells.

CXCR4, a member of the family of chemokine-activated G protein-coupled receptors, is widely expressed in immune response cells. It is involved in both cancer development and progression as well as viral infection, notably by HIV-1. A variety of methods, including structural information, have suggested that the receptor may exist as a dimer or an oligomer. However, the mechanistic details surrounding receptor oligomerization and its potential dynamic regulation remain unclear. Using both biochemical and biophysical means, we confirm that CXCR4 can exist as a mixture of monomers, dimers, and higher-order oligomers in cell membranes and show that oligomeric structure becomes more complex as receptor expression levels increase. Mutations of CXCR4 residues located at a putative dimerization interface result in monomerization of the receptor. Additionally, binding of the CXCR4 antagonist IT1t-a small drug-like isothiourea derivative-rapidly destabilizes the oligomeric structure, whereas AMD3100, another well-characterized CXCR4 antagonist, does not. Although a mutation that regulates constitutive activity of CXCR4 also results in monomerization of the receptor, binding of IT1t to this variant promotes receptor dimerization. These results provide novel insights into the basal organization of CXCR4 and how antagonist ligands of different chemotypes differentially regulate its oligomerization state.

While the notion that G protein-coupled receptors (GPCRs) associate into homo- and hetero-oligomers has gained more recognition in recent years, a lack of consensus remains among researchers regarding the functional relevance of GPCR oligomerization. A relatively recent technique, Förster resonance energy transfer (FRET) spectrometry, allows for the determination of the oligomeric (or quaternary) structure of proteins in living cells via analysis of efficiency distributions of energy transferred from optically excited fluorescent tags acting as donors of energy to fluorescent tags acting as acceptors of energy and residing within the same oligomer. In this study, we significantly improved the resolution of the FRET-spectrometry approach to detect small differences between the interprotomeric distances among GPCR oligomers with subtle differences in quaternary structures. We then used this approach to study the conformational substates of oligomers of sterile 2 α-factor receptor (Ste2), a class D GPCR found in the yeastSaccharomyces cerevisiaeof mating typea. Ste2 has previously been shown to form tetrameric oligomers at relatively low expression levels (between 11 and 140 molecules/μm2) in the absence of its cognate ligand, the α-factor pheromone. The significantly improved FRET spectrometry technique allowed us to detect multiple distinct quaternary conformational substates of Ste2 oligomers, and to assess how the α-factor ligand altered the proportion of such substates. The ability to determine quaternary structure substates of GPCRs provides exquisite means to elucidate functional relevance of GPCR oligomerization.

Understanding the photophysical properties of fluorescent proteins (FPs), such as emission and absorption spectra, molecular brightness, photostability, and photo-switching, is critical to the development of criteria for their selection as tags for fluorescent-based biological applications. While two-photon excitation imaging techniques have steadily gained popularity - due to comparatively deeper penetration depth, reduced out-of-focus photobleaching, and wide separation between emission spectra and two-photon excitation spectra -, most studies reporting on the photophysical properties of FPs tend to remain focused on single-photon excitation. Here, we report our investigation of the photophysical properties of several commonly used fluorescent proteins using two-photon microscopy with spectral resolution in both excitation and emission. Our measurements indicate that not only the excitation (and sometimes emission) spectra of FPs may be markedly different between single-photon and two-photon excitation, but also their relative brightness and their photo-stability. A good understanding of the photophysical properties of FPs under two-photon excitation is essential for choosing the right tag(s) for a desired experiment.

The resistance of methicillin-resistant Staphylococcus aureus to antibiotics presents serious clinical problems that prompted the need for finding alternative or combination therapies. One such therapy is irradiation with blue light. To determine the alterations in metabolic processes implicated in the observed antimicrobial effects of blue light, we investigated the changes in membrane potential and the presence of free-radical-producing photo-acceptor molecules. Bacterial cultures irradiated with one or two doses of 405nm laser light (each consisting of 121J/cm2) were imaged with spectrally resolved laser-scanning microscopes to detect endogenous fluorescent species as well as the voltage sensitive dye 3,3'-Diethyloxacarbocyanine iodide. The endogenous fluorescence indicated the presence of photosensitizers (i.e., porphyrins, NADH, FAD) in the cells, while the exogenous signal allowed us to monitor rapid changes in transmembrane potential following treatment with light. The changes were drastic within the first 5min after irradiation with the first dose and continued slowly after the second irradiation. These results suggest that the early antimicrobial activity of blue light results from alteration of membrane integrity with a consequent decrease in membrane polarization and rapid alteration of vital cellular functions. The observation of an early antimicrobial activity of light is very encouraging, as it suggests that treatment does not necessarily have to be administered over a long period of time.

Annibale and Lohse have recently suggested a way1 in which the two-dimensional fluorescence intensity fluctuation (2D FIF) spectrometry2 may be further refined. Their main suggestion is to include a step in the analysis process where a case-by-case inspection of individual regions of interest of a membrane allows for selection of portions of the membrane which are “as homogenous as possible” and thereby exclude intensity spots potentially related to other sub-cellular structures. By incorporating that proposal into an objective and reproducible algorithm, here we show that 2D FIF has a built-in capability to automatically filter out such contributions, and that further removal of inhomogeneities does not alter the final results.

Current methods for determining membrane protein association in cells are either very time consuming, require complicated procedures, or lack the sensitivity needed to assess the effect of concentration or ligand binding on the observed oligomerization. To overcome these limitations, we provide a detailed protocol for quantifying the relative abundance and stability of oligomers of differing sizes using two-dimensional fluorescence intensity fluctuation (2D-FIF) spectrometry. The approach can be implemented using a standard laser scanning fluorescence microscope along with custom written software for image analysis. This method may be applied to evaluate the extent of oligomerization as a function of receptor concentration and is particularly suited to assess the effects of agonists and antagonists on the oligomeric size of membrane receptors of interest.

Using radiofrequency dielectric spectroscopy, we have investigated the impact of the interaction between a G protein-coupled receptor (GPCR), the sterile2 α-factor receptor protein (Ste2), and its cognate agonist ligand, the α-factor pheromone, on the dielectric properties of the plasma membrane in living yeast cells (Saccharomyces cerevisiae). The dielectric properties of a cell suspension containing a saturating concentration of α-factor were measured over the frequency range 40Hz–110 MHz and compared to the behavior of a similarly prepared suspension of cells in the absence of α-factor. A spherical three-shell model was used to determine the electrical phase parameters for the yeast cells in both types of suspensions. The relative permittivity of the plasma membrane showed a significant increase after exposure to α-factor (by 0.06 ± 0.05). The equivalent experiment performed on yeast cells lacking the ability to express Ste2 showed no change in plasma membrane permittivity. Interestingly, a large change also occurred to the electrical properties of the cellular interior after the addition of α-factor to the cell suspending medium, whether or not the cells were expressing Ste2. We present a number of different complementary experiments performed on the yeast to support these dielectric data and interpret the results in terms of specific cellular reactions to the presence of α-factor.

Resonance energy transfer (RET) and fluorescence fluctuation spectroscopies (FFS) are powerful fluorescence-based techniques for quantifying the self-association of membrane receptors within oligomeric complexes in living cells. However, RET spectrometry's ability to extract information on the detailed quaternary structure of oligomers sometimes rests on assumptions regarding the relative abundances of oligomers of different sizes, while FFS techniques may provide oligomer size information but not quaternary structure details, as they lack a probe for inter-molecular distances. In this report, we introduce a method which we termed 'intensity fluctuations and resonance energy transfer' (IFRET), which combines analysis of donor and acceptor intensity fluctuations with RET efficiency determination. Because the three measured quantities each have a unique dependence on the acceptor mole fraction (X A ), simultaneous global fitting of all three dramatically reduces ambiguity in the data fitting and choice of the most appropriate fitting model. We demonstrate the effectiveness of the method on simulated brightness and RET efficiency data incorporating mixtures of monomers, dimers, and tetramers and show that IFRET analysis provides a major improvement in both identifying the correct quaternary structure model and extracting the relative abundances of the monomers, dimers, and tetramers. Conceivably, the enhanced resolution of IFRET could potentially provide insight into the functional significance of receptor oligomerization in the presence and absence of cognate ligands.

Current technologies for probing membrane protein association and stability in cells are either very laborious or lack the bandwidth needed for fully quantitative analysis. Here we introduce a platform, termedone-ortwo-dimensional fluorescence intensity fluctuation spectrometry, for determining the identity, abundance, and stability of oligomers of differing sizes. The sensitivity of this approach is demonstrated by using monomers and oligomers of known sizes in both solutions and cell membranes. The analysis was extended to uncover the oligomeric states and their stability for both the epidermal growth factor receptor, a receptor tyrosine kinase, and the G protein-coupled secretin receptor. In both cases, agonist ligand binding shifted the equilibrium from monomers or dimers to rather large oligomers. Our method can be used in conjunction with a variety of light-based microscopy techniques, is several orders of magnitude faster than current approaches, and is scalable for high-throughput analyses.

Here, we introduce fluorescence intensity fluctuation spectrometry for determining the identity, abundance and stability of protein oligomers. This approach was tested on monomers and oligomers of known sizes and was used to uncover the oligomeric states of the epidermal growth factor receptor and the secretin receptor in the presence and absence of their agonist ligands. This method is fast and is scalable for high-throughput screening of drugs targeting protein-protein interactions.

Here we introduce the fully quantified spectral imaging (FSI) method as a new tool to probe the stoichiometry and stability of protein complexes in biological membranes. The FSI method yields two dimensional membrane concentrations and FRET efficiencies in native plasma membranes. It can be used to characterize the association of membrane proteins: to differentiate between monomers, dimers, or oligomers, to produce binding (association) curves, and to measure the free energies of association in the membrane. We use the FSI method to study the lateral interactions of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), a member of the receptor tyrosine kinase (RTK) superfamily, in plasma membranes, in vivo. The knowledge gained through the use of the new method challenges the current understanding of VEGFR2 signaling.

Multiphoton micro-spectroscopy, employing diffraction optics and electron-multiplying CCD (EMCCD) cameras, is a suitable method for determining protein complex stoichiometry, quaternary structure, and spatial distribution in living cells using Förster resonance energy transfer (FRET) imaging. The method provides highly resolved spectra of molecules or molecular complexes at each image pixel, and it does so on a timescale shorter than that of molecular diffusion, which scrambles the spectral information. Acquisition of an entire spectrally resolved image, however, is slower than that of broad-bandwidth microscopes because it takes longer times to collect the same number of photons at each emission wavelength as in a broad bandwidth. Here, we demonstrate an optical micro-spectroscopic scheme that employs a laser beam shaped into a line to excite in parallel multiple sample voxels. The method presents dramatically increased sensitivity and/or acquisition speed and, at the same time, has excellent spatial and spectral resolution, similar to point-scan configurations. When applied to FRET imaging using an oligomeric FRET construct expressed in living cells and consisting of a FRET acceptor linked to three donors, the technique based on line-shaped excitation provides higher accuracy compared to the point-scan approach, and it reduces artifacts caused by photobleaching and other undesired photophysical effects.

Transmembrane proteins known as G protein-coupled receptors (GPCRs) have been shown to form functional homo- or hetero-oligomeric complexes, although agreement has been slow to emerge on whether homo-oligomerization plays functional roles. Here we introduce a platform to determine the identity and abundance of differing quaternary structures formed by GPCRs in living cells following changes in environmental conditions, such as changes in concentrations. The method capitalizes on the intrinsic capability of FRET spectrometry to extract oligomer geometrical information from distributions of FRET efficiencies (or FRET spectrograms) determined from pixel-level imaging of cells, combined with the ability of the statistical ensemble approaches to FRET to probe the proportion of different quaternary structures (such as dimers, rhombus or parallelogram shaped tetramers, etc.) from averages over entire cells. Our approach revealed that the yeast pheromone receptor Ste2 forms predominantly tetramers at average expression levels of 2 to 25 molecules per pixel (2.8 · 10 − 6 to 3.5 · 10 − 5 molecules/nm 2 ), and a mixture of tetramers and octamers at expression levels of 25–100 molecules per pixel (3.5 · 10 − 5 to 1.4 · 10 − 4 molecules/nm 2 ). Ste2 is a class D GPCR found in the yeast Saccharomyces cerevisiae of the mating type a , and binds the pheromone α-factor secreted by cells of the mating type α . Such investigations may inform development of antifungal therapies targeting oligomers of pheromone receptors. The proposed FRET imaging platform may be used to determine the quaternary structure sub-states and stoichiometry of any GPCR and, indeed, any membrane protein in living cells. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

Rhodopsin is a prototypical G-protein-coupled receptor (GPCR) that initiates phototransduction in the retina. The receptor consists of the apoprotein opsin covalently linked to the inverse agonist 11-cis retinal. Rhodopsin and opsin have been shown to form oligomers within the outer segment disc membranes of rod photoreceptor cells. However, the physiological relevance of the observed oligomers has been questioned since observations were made on samples prepared from the retina at low temperatures. To investigate the oligomeric status of opsin in live cells at body temperatures, we utilized a novel approach called Förster resonance energy transfer spectrometry, which previously has allowed the determination of the stoichiometry and geometry (i.e. quaternary structure) of various GPCRs. In the current study, we have extended the method to additionally determine whether or not a mixture of oligomeric forms of opsin exists and in what proportion. The application of this improved method revealed that opsin expressed in live Chinese hamster ovary (CHO) cells at 37°C exists as oligomers of various sizes. At lower concentrations, opsin existed in an equilibrium of dimers and tetramers. The tetramers were in the shape of a near-rhombus. At higher concentrations of the receptor, higher-order oligomers began to form. Thus, a mixture of different oligomeric forms of opsin is present in the membrane of live CHO cells and oligomerization occurs in a concentration-dependent manner. The general principles underlying the concentration-dependent oligomerization of opsin may be universal and apply to other GPCRs as well.

The Maxwell–Wagner theory of interfacial polarization treats dielectric particles suspended in homogeneous media and subjected to alternating fields as systems of independent induced dipoles, and therefore predicts simple Debye-type dispersion in which both permittivity and conductivity vary between low- and high-frequency plateaus. Unfortunately, Maxwell–Wagner theory cannot replicate its own success when applied to more concentrated and structured (i.e., non-random) systems of cells such as tissues. The effective medium theory, devised to incorporate contributions of the induced dipoles to the average far-field, brought about some improvement, but the general shape of the dielectric spectra remained that of a simple Debye – in contrast with experimental data. Supracellular organization of tissues induces non-Debye behavior due to near-field interactions between neighboring cells. Specifically, biological cells may perturb each other’s electrical environment via dipolar (multipolar) fields or conductance bridges (such as pores and gap junctions) between cytoplasms and across cell membranes. We substantiate this hypothesis by presenting comparatively data from yeast and red blood cell suspensions, blood forming reversible supra-cellular structures, called ‘rouleaux’, breast tissues, as well as from brain treated with anesthetics that are known to affect the gap-junctional connectivity between cells.

Resonant energy transfer from an optically excited donor molecule to a non-excited acceptor molecule residing nearby is widely used to detect molecular interactions in living cells. To date, resonant energy transfer has been used to obtain stoichiometric information, such as the number of proteins forming a complex, for a handful of proteins, but only after performing sequential scans of the emission wavelengths, excitation wavelengths, or sometimes both. During this lengthy process of measurement, the molecular makeup of a cellular region may change, limiting the applicability of resonant energy transfer to the determination of cellular averages. Here, we demonstrate a method for the determination of protein complex size, configuration, and spatial distribution in single living cells. It relies on a spectrally resolved two-photon microscope, a simple but competent theory, and a judicious selection of fluorescent tags. This approach eventually may lead to tracking the dynamics of individual molecular complexes inside living cells. The combination of spectrally resolved two-photon microscopy, fluorescent tags and appropriate theory makes it possible to determine the complex size, configuration and spatial distribution of proteins in single living cells. The findings made could lead to ways of tracking the cellular dynamics of individual molecular complexes.

We report an improved method for production of microfluidic device masters using two-photon photopolymerization of SU-8 negative photoresist, which relies on a two-photon microscope (TPM) commonly used in imaging of biological samples. The device masters serve as negative relief structures for polydimethylsiloxane-based microfluidic devices. We observed that not only did the two-photon excitation of the SU-8 photoresist initiate crosslinking of the material in the region of the focus of the near-infrared laser beam (as expected) but it also resulted in emission of fluorescence in the visible range. The detected emission of SU-8 photoresist undergoing two-photon excitation displayed a strong correlation with the size of the polymerized objects produced during the exposure; this allowed the progress of the microfluidic master production process to be monitored in real-time. We demonstrate the use of the fluorescence detection during two-photon photopolymerization in the production of microfluidic devices, which were designed to trap individual yeast cells to be imaged with the same TPM used for microfluidic master writing.

Spurious contributions from electrode polarization (EP) are a major nuisance in dielectric measurements of biological tissues and hamper accurate determination of tissue properties in the audio/radiofrequencies. Various electrode geometries and/or treatments have been employed traditionally to reduce EP contributions, although none succeeded to completely remove EP from measurements on tissues for all practical frequency ranges. A method of correction for contributions of EP to the dielectric properties of tissues is proposed. The method is based on modeling the electrode impedance with suitable functions and on the observation that certain parameters are only dependent on electrodes properties and can thus be determined separately. The method is tested on various samples with known properties, and its usefulness is demonstrated with samples of normal and cancerous human female breast tissue. It is observed that the dielectric properties of the tissues over the frequency range 40 Hz-100 MHz are significantly different among different types of breast tissue. This observation is used further to demonstrate that, by scanning the tip of the measuring dielectric probe (with modest spatial resolution) across a sample of excised breast tissue, significant variations in the electrical properties are detected at a position where a tumor is located. This study shows that dielectric spectroscopy has the potential to offer a viable alternative to the current methods for detection of breast cancer in vivo.

Pseudomonas aeruginosa is a pathogenic Gram-negative bacterium that affects patients with cystic fibrosis and immunocompromised individuals. This bacterium coexpresses two unique forms of lipopolysaccharides (LPSs) on its surface, the A- and B-band LPS, which are among the main virulence factors that contribute to its pathogenicity. The polysaccharides in A-band LPSs are synthesized in the cytoplasm and translocated into the periplasm via an ATP-binding cassette (ABC) transporter consisting of a transmembrane protein, Wzm, and a cytoplasmic nucleotide-binding protein, Wzt. Most of the biochemical studies of A-band PSs in Pseudomonas aeruginosa are focused on the stages of the synthesis and ligation of PS, leaving the export stage involving the ABC transporter mostly unexplored. This difficulty is compounded by the fact that the subunit composition and structure of this bi-component ABC transporter are still unknown. Here we propose a simple but powerful method, based on Forster Resonance Energy Transfer (FRET) and optical micro-spectroscopy technology, to probe the structure of dynamic (as opposed to static) protein complexes in living cells. We use this method to determine the association stoichiometry and quaternary structure of the Wzm–Wzt complex in living cells. It is found that Wzt forms a rhombus-shaped homo-tetramer which becomes a square upon co-expression with Wzm, and that Wzm forms a square-shaped homo-tetramer both in the presence and absence of Wzt. Based on these results, we propose a structural model for the double-tetramer complex formed by the bi-component ABC transporter in living cells. An understanding of the structure and behavior of this ABC transporter will help develop antibiotics targeting the biosynthesis of the A-band LPS endotoxin.

Advances in two-photon microscopy with spectral resolution (TPM-SR) and the development of a simple theory of Forster Resonance Energy Tran sfer (FRET) for single molecular complexes recently lead to the development of a novel method for the determination of structure and localization in living cells of membrane protein complexes (Raicu et al., Nature Photon. , 3, 2009). An appealing feature of this method is its ability to provide such important information while being unaffected by spurious signals originating from stochastic FRET (Singh and Raicu, Biophys. J., 98 , 2010). We will present the results obtained from our recent studies of trimeric FRET calibration standards expressed in the cytoplasm of Chinese hamster ovary (CHO) cells, as well as a model G protein-coupled receptor expressed in the membrane of yeast. Emphasis will be placed on the measurement and analysis of single-molecular-complex FRET data for determination of the quaternary structure of some proteins (or the protein complex structure). Keywords: Forster (Fluorescence) Resonance Energy Transfer (FRET); two-photon microscopy; Chinese hamster ovary (CHO) cells; G protein-coupled receptor (GPCR); spectral resolution; sterile two alpha-factor receptor.

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. Forster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 α-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

Resonance Energy Transfer (RET) between a donor molecule in an electronically excited state and an acceptor molecule in close proximity has been frequently utilized for studies of protein-protein interactions in living cells. Typically, the cell under study is scanned a number of times in order to accumulate enough spectral information to accurately determine the RET efficiency for each region of interest within the cell. However, the composition of these regions may change during the course of the acquisition period, limiting the spatial determination of the RET efficiency to an average over entire cells. By means of a novel spectrally resolved two-photon microscope, we were able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of RET efficiencies throughout the cell is calculated. By applying a simple theory of RET in oligomeric complexes to the experimentally obtained distribution of RET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. This presentation will describe our experimental setup and data analysis procedure, as well as an application of the method to the determination of RET efficiencies throughout yeast cells ( S. cerevisiae ) expressing a G-protein-coupled receptor, Sterile 2 α factor protein (Ste2p), in the presence and absence of α-factor - a yeast mating pheromone.

Resonant Energy Transfer (RET) from an optically excited molecule to a non-excited molecule residing nearby has been used to detect molecular interactions in living cells. Information such as the number of proteins forming a molecular complex has been obtained so far for a handful of proteins, but only after exposing the samples sequentially to at least two different excitation wavelengths. Changes in the molecular makeup of a cellular region occurring during this lengthy process of measurement has limited the applicability of RET to determination of cellular averages. We developed a method for imaging protein complex distribution in living cells with sub-cellular spatial resolution, which relies on a spectrally-resolved two-photon microscope. The use of diffractive optics in a non-descanned configuration allows acquisition of a full set of spectrally-resolved images after only one complete scan of the excitation beam. This presentation will briefly describe our basic experimental setup and a simple theory of RET in oligomeric complexes, and it will review our recent results on determination of the geometry and size of oligomeric complexes of several proteins in yeast as well as in mammalian cells. This method basically transforms RET into a method for performing veritable structural determinations of protein complexes in vivo.

Modelocked Ti:Sapphire lasers are widely used in two-photon microscopes (TPM), partly due to their tunability over a broad range of wavelengths (between 700 nm and 1000 nm). Many biophysical applications, including quantitative Forster Resonance Energy Transfer (FRET) and photoswitching of fluorescent proteins between dark and bright states, require wavelength tuning without optical realignment, which is not easily done in tunable Ti:Sapphire lasers. In addition, for studies of dynamics in biological systems the time required for tuning the excitation should be commensurate with the shortest of the time scales of the processes investigated. A set-up in which a modelocked Ti:Sapphire oscillator providing broad-bandwidth (i.e., short) pulses with fixed center wavelength is coupled to a pulse shaper incorporating a spatial light modulator placed at the Fourier plane of a zero-dispersion two-grating setup, represents a faster alternative to the tunable laser. A pulse shaping system and a TPM with spectral resolution allowed us to acquire two-photon excitation and emission spectra of fluorescent molecules in single living cells. Such spectra may be exploited for mapping intracellular pH and for quantitative studies of protein localization and interactions in vivo.

We have investigated the origin of the dielectric response of the plasma membrane of living yeast cells ( Saccharomyces cerevisiae ) by using radiofrequency dielectric spectroscopy. The cells were genetically engineered to overexpress in the membrane of yeast cells a G protein-coupled receptor – the Sterile2-α factor receptor protein (Ste2p) – fused to the green fluorescent protein (GFP). Presence of the Ste2-GFP proteins in the plasma membrane was confirmed by exciting the cells at 476 nm and observing with a confocal microscope the emission characteristic of the GFP from individual cells. The dielectric behavior of cells suspended in KCl solution was analyzed over the frequency range 40 Hz–110 MHz and compared to the behavior of control cells that lacked the ability to express Ste2p. A two-shell electrical cell model was used to fit the data starting from known structural parameters and adjustable electrical phase parameters. The best-fit value for the relative permittivity of the plasma membrane showed no significant difference between cells expressing Ste2p (1.63 ± 0.11) and the control cells (1.75 ± 0.16). This result confirmed earlier predictions that the dielectric properties of the plasma membrane in the radiofrequency range mostly reflect the properties of the hydrophobic layer of the membrane, which is populated by the hydrocarbon tails of the phospholipids and hydrophobic segments of integral membrane proteins. We discuss ways by which dielectric spectroscopy can be improved to be used for tag-free detection of proteins on the membrane.

Forster Resonance Energy Transfer (FRET) from an electronically excited donor to an acceptor molecule is used to quantify the extent of interactions between molecules. For example, FRET can be utilized to determine how proteins form complexes by tagging them differentially with donor and acceptor fluorophores. Recent advances in the FRET theory combined with a novel spectrally resolved two-photon microscope have strengthened the effectiveness of the FRET technique and have enabled us to determine the size and structure of protein oligomers in living cells (Raicu et al., Nature Photonics, 3, 2009). In an ongoing research project in our lab, we are are probing the movement of protomers in an oligomer by appropriately tagging and systematically altering a fluorophore position within each protomer. In the work described here, we show the application of this method in yeast cells (S. cerevisiae) that express the sterile 2 alpha factor protein (Ste2p, a G protein-coupled receptor) tagged with two different variants of the green fluorescent protein (GFP). Previously, we showed by tagging GFP2/YPF (donor/acceptor fluorophores) variants at the C-terminus of Ste2p that Ste2p is self-assembled into a rhombus shaped tetramer. The measured FRET efficiencies of the Ste2p oligomers, which were tagged with fluorophores at various locations in the Ste2p amino acid sequence, were calibrated against FRET reference standards in order to extract information regarding the relative orientations of the fluorescent tags. The effect of an agonist, the yeast mating pheromone alpha factor, on the measured FRET efficiencies was quantified and compared for the various fluorescent tag locations to reveal information regarding the relative movement of the Ste2p protomer segments upon binding of the agonist.

Forster Resonance Energy Transfer (FRET) between donor and acceptor molecules is widely used for detection of molecular interactions. When donor and acceptor tags are fused to proteins of interest, FRET may be used to probe whether the tagged proteins form functional complexes in living cells. Typically, the cell under study is scanned at several times in order to accumulate enough spectral information to determine the FRET efficiency for each region of interest within the cell. However, diffusion as well as biochemical reactions may cause the molecular make-up of the regions of interest to change during the course of data acquisition. For a long time, this has dramatically limited the information content of FRET imaging. Advances in theory and optical instrumentation recently lead to the development of a FRET technique that avoids the problems caused by molecular diffusion and enabled us to determine the stoichiometry, structure, and localization in living cells of membrane protein complexes (Raicu et al., Nature Photonics, 3, 2009). This presentation will review the results obtained from our recent studies of oligomeric complexes of some G protein-coupled receptors (GPCRs) in vivo, both, in the presence and in the absence of natural as well synthetic ligands.

We have determined the oligomeric size and configuration of fluorophore-tagged M1, M2, β1, and β2 receptors in the plasma membrane of Chinese hamster ovary (CHO) cells by examining the distribution of FRET efficiencies measured at the level of single pixels. The receptors were fused at the N-terminus to enhanced green or yellow fluorescent protein, and complementary pairs were co-expressed at different ratios of donor to acceptor (i.e., eGFP2-M1 and eYFP-M1, eGFP2-M2 and eYFP-M2, eGFP2-β1 and eYFP-β1, or eGFP2-β2 and eYFP-β2). Pixel-level emission spectra were recorded from images captured in a single plane; the relative contribution of each fluorophore then was determined by spectral deconvolution and used to calculate the corresponding apparent FRET efficiency. The distributions of efficiencies from 20 cells were analyzed in concert as a sum of Gaussians, with the number of components (n) and the relationships among the means taken as predicted for a dimer (n = 1), a triangular trimer (n = 2), and a tetramer configured as a square (n = 3) and a rhombus (n = 5). Distributions from each of the four receptors required 5 Gaussians, the means of which were related in the manner predicted for a rhombus. The inverse agonists atropine and timolol were without effect on the number of components or the relationship among the means detected for the M2 muscarinic receptor and the β2 adrenergic receptor, respectively. Homo-oligomers of the M1, M2, β1, and β2 receptors therefore appear to be rhombic tetramers in CHO cells. The configuration is unaffected by inverse agonists, at least in the case of the M2 and β2 receptors.

Forster Resonance Energy Transfer (FRET) is a process in which a donor (D) in the excited state transfers its energy nonradiatively to an acceptor (A) in the ground state. The underlying theory has been confirmed countless times, particularly with regard to the dependence of the FRET efficiency on the sixth power of the distance between D and A. In contrast, a complete FRET theory for multiple donors and acceptors in oligomeric complexes has been developed only recently (Raicu, 2007, J. Biol. Phys. 33:109-127), in parallel with technology of sufficient accuracy for tests in living cells (Raicu et al., 2009, Nature Photon. 3:107-113). This novel approach now has been applied to linked fluorescent proteins located in the cytoplasm and at the plasma membrane. The cytoplasmic probes were fused combinations of a donor (Cerulean, C), an acceptor (Venus, V), and a chromophore-deficient, Venus-like molecule that cannot absorb or transfer energy (Amber, A) (Koushik et al., 2009, PLoS ONE 4(11):e8031): namely, ACVA, ACAV, VCAA, and VCVV. The membrane-bound probes were fused dimers and trimers of eGFP2 (G2) and eYFP (Y): namely, G2Y, YG2, G2YG2, and YG2Y. According to the theory (Raicu, 2007), the FRET efficiency of a tetramer such as VCVV can be predicted from that of analogues that contain a single acceptor (e.g., ACVA, ACAV, VCAA); also, the apparent FRET efficiency of a trimer such as G2YG2 or YG2Y can be predicted from the pair-wise efficiency that corresponds to that of dimers such as G2Y and YG2. These predictions have been confirmed for FRET efficiencies measured by means of two-photon microspectroscopy (Raicu et al., 2009), in accord with the theory and underlying assumptions for FRET within multimers.

G protein-coupled receptors are known to form oligomers, but estimates of their size range from dimers to large arrays. We therefore have determined the oligomeric size of fluorophore-tagged M2 and β2 receptors in the plasma membrane of Chinese hamster ovary cells by examining the distribution of FRET efficiencies measured at the level of single pixels. Each receptor was fused at its N-terminus to enhanced green or yellow fluorescent protein and co-expressed as the complementary pair (EGFP2-M2 and EYFP-M2, or EGFP2-β2 and EYFP-β2). Pixel-level emission spectra were recorded from images captured in a single plane; the relative contribution of each fluorophore then was determined by spectral deconvolution and used to calculate the corresponding FRET efficiency. The distribution of efficiencies from each cell was analyzed as a sum of Gaussians. The number of Gaussians and the numeric relationship among the corresponding means (E) is determined by the number of combinatorial arrangements of FRET-productive pairs within a two-dimensional oligomer as predicted by the binomial theorem. A dimer will reveal a single efficiency or Gaussian, and a triangular trimer will reveal two; a square tetramer will reveal three efficiencies, and a rhombus will reveal five. Both the M2 receptor and the β2 receptor required five Gaussians to describe the distributions of efficiencies from several cells taken together. In each case, an efficiency (E) identified as the pair-wise efficiency (Ep) was related to the other four efficiencies in the manner predicted for a rhombus.