Search

Förster resonance energy transfer (FRET) is a widely used technique for nanoscale molecular distance measurements, which makes FRET ideal for studying protein interactions and quaternary structure of protein complexes. In this work, we were interested in how photobleaching of donor and acceptor molecules affects the FRET results under various excitation conditions. We conducted a systematic study, under two-photon excitation, of the effects of the excitation power and the choice of excitation wavelengths upon the measured FRET efficiencies of multiplex protein constructs, consisting of one donor (D) and two acceptors (A) or one acceptor and a non-fluorescent tag (N), using both the kinetic theory of FRET and numerical simulations under given excitation conditions. We found that under low excitation power and properly chosen excitation wavelengths the relationship between the FRET efficiency of a trimeric construct ADA agrees within 2% with the FRET efficiency computed (via the kinetic theory of FRET in the absence of photobleaching) from two dimeric constructs ADN and NDA. By contrast, at higher excitation powers the FRET efficiencies changed significantly due to the photobleaching of both the donor (through direct excitation) and the acceptor (mostly through FRET-induced excitation). Based on these results and numerical simulations using a simple but competent algorithm, we developed guidelines for choosing appropriate experimental conditions for reliable FRET measurements, as well as for interpreting the results of existing experiments using different excitation schemes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The functional significance of the interactions between proteins in living cells to form short-lived quaternary structures cannot be overemphasized. Yet, quaternary structure information is not captured by current methods, neither can those methods determine structure within living cells. The dynamic versatility, abundance, and functional diversity of G protein-coupled receptors (GPCRs) pose myriad challenges to existing technologies but also present these proteins as the ideal testbed for new technologies to investigate the complex inter-regulation of receptor-ligand, receptor-receptor, and receptor-downstream effector interfaces in living cells. Here, we present development and use of a novel method capable of overcoming existing challenges by combining distributions (or spectrograms) of FRET efficiencies from populations of fluorescently tagged proteins associating into oligomeric complexes in live cells with diffusion-like trajectories of FRET donors and acceptors obtained from molecular dynamics (MD) simulations. Our approach provides an atom-level picture of the binding interfaces within oligomers of the human secretin receptor (hSecR) in live cells and allows for extraction of mechanistic insights into the function of GPCRs oligomerization. This FRET-MD spectrometry approach is a robust platform for investigating protein-protein binding mechanisms and opens a new avenue for investigating stable as well as fleeting quaternary structures of any membrane proteins in living cells.

Förster resonance energy transfer (FRET) spectrometry is a method for determining the quaternary structure of protein oligomers from distributions of FRET efficiencies that are drawn from pixels of fluorescence images of cells expressing the proteins of interest. FRET spectrometry protocols currently rely on obtaining spectrally resolved fluorescence data from intensity-based experiments. Another imaging method, fluorescence lifetime imaging microscopy (FLIM), is a widely used alternative to compute FRET efficiencies for each pixel in an image from the reduction of the fluorescence lifetime of the donors caused by FRET. In FLIM studies of oligomers with different proportions of donors and acceptors, the donor lifetimes may be obtained by fitting the temporally resolved fluorescence decay data with a predetermined number of exponential decay curves. However, this requires knowledge of the number and the relative arrangement of the fluorescent proteins in the sample, which is precisely the goal of FRET spectrometry, thus creating a conundrum that has prevented users of FLIM instruments from performing FRET spectrometry. Here, we describe an attempt to implement FRET spectrometry on temporally resolved fluorescence microscopes by using an integration-based method of computing the FRET efficiency from fluorescence decay curves. This method, which we dubbed time-integrated FRET (or tiFRET), was tested on oligomeric fluorescent protein constructs expressed in the cytoplasm of living cells. The present results show that tiFRET is a promising way of implementing FRET spectrometry and suggest potential instrument adjustments for increasing accuracy and resolution in this kind of study.

Fluorescence emission is common in plants. While fluorescence microscopy has been widely used to study living plants, its application in quantifying the fluorescence of fossil plants has been limited. Fossil plant fluorescence, from original fluorophores or formed during fossilization, can offer valuable insights into fluorescence in ancient plants and fossilization processes. In this work, we utilize two-photon fluorescence microspectroscopy to spatially and spectrally resolve the fluorescence emitted by amber-embedded plants, leaf compressions, and silicified wood. The advanced micro-spectroscope utilized, with its pixel-level spectral resolution and line-scan excitation capabilities, allows us to collect comprehensive excitation and emission spectra with high sensitivity and minimal laser damage to the specimens. By applying linear spectral unmixing to the spectrally resolved fluorescence images, we can differentiate between (a) the matrix and (b) the materials that comprise the fossil. Our analysis suggests that the latter correspond to durable tissues such as lignin and cellulose. Additionally, we observe potential signals from chlorophyll derivatives/tannins, although minerals may have contributed to this. This research opens doors to exploring ancient ecosystems and understanding the ecological roles of fluorescence in plants throughout time. Furthermore, the protocols developed herein can also be applied to analyze non-plant fossils and biological specimens., (© 2024. The Author(s).)

Suberin, a complex biopolymer, forms a water- and gas-insoluble barrier that protects the inner tissues of plants. It is abundant in tree bark, particularly in the cork oak Quercus suber. Anatomically, fossil bark has been described since the Devonian. However, its distinctive constituent suberin has not yet been reported from the fossil record. Here we present unambiguous chemical evidence for intact suberin from the bark of a middle Eocene monkeyhair tree from Geiseltal, eastern Germany. High-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) detected constituents of suberin in the outer layer the fossil monkeyhair tree, which confirms previous morphological interpretation of this tissue as bark, and chemically differentiates this layer from the two tissues of the inner layer. Notably, this is the first study with compelling chemical evidence for suberin in fossil bark. Fluorescence microspectroscopy additionally supports the presence of suberin. Fossilization conditions in the Eocene Geiseltal deposit were likely mild, with low moisture and temperatures, contributing to the remarkable preservation of bark and inner laticifer mats of the monkeyhair trees growing there 45 million years ago., (© 2024. The Author(s).)

Recent advances in fluorescence-based microscopy techniques, such as single molecule fluorescence, Förster resonance energy transfer (FRET), fluorescence intensity fluctuations analysis, and super-resolution microscopy have expanded our ability to study proteins in greater detail within their native cellular environment and to investigate the roles that protein interactions play in biological functions, such as inter- and intracellular signaling and cargo transport. In this Perspective, we provide an up-to-date overview of the current state of the art in fluorescence-based detection of proteins and their interactions in living cells with an emphasis on recent developments that have facilitated the characterization of the spatial and temporal organization of proteins into oligomeric complexes in the presence and absence of natural and artificial ligands. Further advancements in this field will only deepen our understanding of the underlying mechanisms of biological processes and help develop new therapeutic targets.

The receptor tyrosine kinase (RTK) EphA2 is expressed in epithelial and endothelial cells and controls the assembly of cell-cell junctions. EphA2 has also been implicated in many diseases, including cancer. Unlike most RTKs, which signal predominantly as dimers, EphA2 readily forms high-order oligomers upon ligand binding. Here, we investigated if a correlation exists between EphA2 signaling properties and the size of the EphA2 oligomers induced by multiple ligands, including the widely used ephrinA1-Fc ligand, the soluble monomeric m-ephrinA1, and novel engineered peptide ligands. We used fluorescence intensity fluctuation (FIF) spectrometry to characterize the EphA2 oligomer populations induced by the different ligands. Interestingly, we found that different monomeric and dimeric ligands induce EphA2 oligomers with widely different size distributions. Our comparison of FIF brightness distribution parameters and EphA2 signaling parameters reveals that the efficacy of EphA2 phosphorylation on tyrosine 588, an autophosphorylation response contributing to EphA2 activation, correlates with EphA2 mean oligomer size. However, we found that other characteristics, such as the efficacy of AKT inhibition and ligand bias coefficients, appear to be independent of EphA2 oligomer size. Taken together, this work highlights the utility of FIF in RTK signaling research and demonstrates a quantitative correlation between the architecture of EphA2 signaling complexes and signaling features., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Knowledge of the electrical potentials within different compartments of a biological cell induced by applied alternating fields is needed for assessing the effects of electromagnetic radiation on cells, understanding electroporation and other electric field-induced effects, and deriving expressions for the complex permittivity of suspensions of cells. In the work presented in this paper, closed-form analytical expressions have been derived for the electrical potentials within different layers of an inhomogeneous particle consisting of four different dielectric layers and suspended in a homogeneous medium. Those expressions have been used to derive, for the case of a realistic model of a cell containing a large concentric organelle, expressions for the transmembrane potentials (at cell and organelle level) and electric fields within the cell compartments induced by applied fields. The results of the present theoretical model indicate points of departure between the present and previous theoretical models. The present theory also confirms the validity of the equivalence approach introduced by Irimajiri and co-workers for computing the complex permittivity for suspensions of multi-shelled particles. In addition, it shows that the electric field is amplified at the level of the cell and organelle membranes, but not within other cell compartments., Competing Interests: Declaration of Competing Interest The author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (© 2022 Wiley Periodicals LLC.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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.

The emergence of an epidemic evokes the need to monitor its spread and assess and validate any mitigation measures enacted by governments and administrative bodies in real time. We present here a method based on previous models of relaxation in fractal structures to observe and quantify this spread and the response of affected populations and governing bodies, and apply it to COVID-19 as a case study. This method provides means to simultaneously track in real time quantities such as the mortality and the recovery rates as well as the number of new infections caused by an infected person. With sufficient data, this method enables thorough monitoring and assessment of an epidemic without ad-hoc assumptions regarding the evolution of the pandemic in the future., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 Elsevier B.V. All rights reserved.)

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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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 Saccharomyces cerevisiae of mating type a . Ste2 has previously been shown to form tetramers at relatively low expression levels (11 to 140 molecules/μm 2 ) 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.

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.

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

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.

Quantitative imaging methods based on Förster resonance energy transfer (FRET) rely on the determination of an apparent FRET efficiency (E app ), as well as donor and acceptor concentrations, to uncover the identity and relative abundance of the supramolecular (or quaternary) structures of associating macromolecules. Theoretical work has provided "structure-based" relationships between E app distributions and the quaternary structure models that underlie them. By contrast, the body of work that predicates the "signal-based" dependence of E app on directly measurable quantities (i.e., fluorescence emission of donors and acceptors) relies largely on plausibility arguments, one of which is the seemingly obvious assumption that the fraction of fluorescent molecules in the ground state pretty nearly equals the total concentration of molecules. In this work, we use the kinetic models of fluorescence in the presence and absence of FRET to rigorously derive useful relationships between E app and measurable fluorescence signals. Analysis of these relationships reveals a few anticipated results and some unexpected explanations for known experimental FRET puzzles, and it provides theoretical foundations for optimizing measurement strategies., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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.

Perturbations in endoplasmic reticulum (ER) homeostasis, a condition termed ER stress, activate the unfolded protein response (UPR), an intracellular network of signaling pathways. Recently, we have shown that protein kinase Kin1 and its paralog, Kin2, in the budding yeast Saccharomyces cerevisiae (orthologs of microtubule affinity-regulating kinase in humans) contribute to the UPR function. These Kin kinases contain a conserved kinase domain and an autoinhibitory kinase-associated 1 (KA1) domain separated by a long undefined domain. Here, we show that Kin1 or Kin2 protein requires minimally a kinase domain and an adjacent kinase extension region (KER) for UPR function. We also show that the functional mini-Kin2 protein is predominantly visualized inside the cells and precipitated with the cellular membrane fraction, suggesting its association with the cellular endomembrane system. Furthermore, we show that transphosphorylation of the Kin1 residue T302 and the analogous Kin2 residue T281 within the activation loop are important for full kinase activity. Collectively, our data suggest that, during ER stress, the Kin kinase domain is released from its autoinhibitory KA1 domain and is activated by transphosphorylation., (Copyright © 2018 American Society for Microbiology.)

Investigations of static or dynamic interactions between proteins or other biological macromolecules in living cells often rely on the use of fluorescent tags with two different colors in conjunction with adequate theoretical descriptions of Förster Resonance Energy Transfer (FRET) and molecular-level micro-spectroscopic technology. One such method based on these general principles is FRET spectrometry, which allows determination of the quaternary structure of biomolecules from cell-level images of the distributions, or spectra of occurrence frequency of FRET efficiencies. Subsequent refinements allowed combining FRET frequency spectra with molecular concentration information, thereby providing the proportion of molecular complexes with various quaternary structures as well as their binding/dissociation energies. In this paper, we build on the mathematical principles underlying FRET spectrometry to propose two new spectrometric methods, which have distinct advantages compared to other methods. One of these methods relies on statistical analysis of color mixing in subpopulations of fluorescently tagged molecules to probe molecular association stoichiometry, while the other exploits the color shift induced by FRET to also derive geometric information in addition to stoichiometry. The appeal of the first method stems from its sheer simplicity, while the strength of the second consists in its ability to provide structural information., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Human cells detect RNA viruses through a set of helicases called RIG-I-like receptors (RLRs) that initiate the interferon response via a mitochondrial signaling complex. Many RNA viruses also encode helicases, which are sometimes covalently linked to proteases that cleave signaling proteins. One unresolved question is how RLRs interact with each other and with viral proteins in cells. This study examined the interactions among the hepatitis C virus (HCV) helicase and RLR helicases in live cells with quantitative microspectroscopic imaging (Q-MSI), a technique that determines FRET efficiency and subcellular donor and acceptor concentrations. HEK293T cells were transfected with various vector combinations to express cyan fluorescent protein (CFP) or YFP fused to either biologically active HCV helicase or one RLR ( i.e. RIG-I, MDA5, or LGP2), expressed in the presence or absence of polyinosinic-polycytidylic acid (poly(I:C)), which elicits RLR accumulation at mitochondria. Q-MSI confirmed previously reported RLR interactions and revealed an interaction between HCV helicase and LGP2. Mitochondria in CFP-RIG-I:YFP-RIG-I cells, CFP-MDA5:YFP-MDA5 cells, and CFP-MDA5:YFP-LGP2 cells had higher FRET efficiencies in the presence of poly(I:C), indicating that RNA causes these proteins to accumulate at mitochondria in higher-order complexes than those formed in the absence of poly(I:C). However, mitochondria in CFP-LGP2:YFP-LGP2 cells had lower FRET signal in the presence of poly(I:C), suggesting that LGP2 oligomers disperse so that LGP2 can bind MDA5. Data support a new model where an LGP2-MDA5 oligomer shuttles NS3 to the mitochondria to block antiviral signaling., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Plants extensively employ leucine-rich repeat receptor-like kinases (LRR-RLKs), the largest family of RLKs, to control a wide range of growth and developmental processes as well as defense responses. To date, only a few direct downstream effectors for LRR-RLKs have been identified. We previously showed that the LRR-RLK EMS1 (EXCESS MICROSPOROCYTES1) and its ligand TPD1 (TAPETUM DETERMINANT1) are required for the differentiation of somatic tapetal cells and reproductive microsporocytes during early anther development in Arabidopsis thaliana Here, we report the identification of β-carbonic anhydrases (βCAs) as the direct downstream targets of EMS1. EMS1 biochemically interacts with βCA proteins. Loss of function of βCA genes caused defective tapetal cell differentiation, while overexpression of βCA1 led to the formation of extra tapetal cells. EMS1 phosphorylates βCA1 at four sites, resulting in increased βCA1 activity. Furthermore, phosphorylation-blocking mutations impaired the function of βCA1 in tapetal cell differentiation; however, a phosphorylation mimic mutation promoted the formation of tapetal cells. βCAs are also involved in pH regulation in tapetal cells. Our findings highlight the role of βCA in controlling cell differentiation and provide insights into the posttranslational modification of carbonic anhydrases via receptor-like kinase-mediated phosphorylation., (© 2017 American Society of Plant Biologists. All rights reserved.)

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/cm 2 ) 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

FRET is an indispensable experimental tool for studying membrane proteins. Currently, two models are available for researchers to determine the oligomerization state of membrane proteins in a static quenching FRET experiment: the model of Veatch and Stryer, derived in 1977, and the kinetic theory-based model for intraoligomeric FRET, derived in 2007. Because of confinement in two dimensions, a substantial amount of FRET is generated by energy transfer between fluorophores located in separate oligomers in the two-dimensional bilayer. This interoligomeric FRET (also known as stochastic, bystander, or proximity FRET) is not accounted for in either model. Here, we use the kinetic theory formalism to describe the dependence of the FRET efficiency measured in an experiment ( i.e. the "total apparent FRET efficiency") on the interoligomeric FRET due to random proximity within the bilayer and the intraoligomeric FRET resulting from protein-protein interactions. We find that data analysis with both models without consideration of the proximity FRET leads to incorrect conclusions about the oligomeric state of the protein. We show that knowledge of the total surface densities of fluorophore-labeled membrane proteins is essential for correctly interpreting the measured total apparent FRET efficiency. We also find that bulk, two-color, static quenching FRET experiments are best suited for the study of monomeric, dimerizing, or dimeric proteins but have limitations in discerning the order of larger oligomers. The theory and methodology described in this work will allow researchers to extract meaningful parameters from static quenching FRET measurements in biological membranes., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cell signaling pathways mediated by leucine-rich repeat receptor-like kinases (LRR-RLKs) are essential for plant growth, development, and defense. The EMS1 (EXCESS MICROSPOROCYTES1) LRR-RLK and its small protein ligand TPD1 (TAPETUM DETERMINANT1) play a fundamental role in somatic and reproductive cell differentiation during early anther development in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether other cell surface molecules serve as coregulators of EMS1. Here, we show that SERK1 (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1) and SERK2 LRR-RLKs act redundantly as coregulatory and physical partners of EMS1. The SERK1/2 genes function in the same genetic pathway as EMS1 in anther development. Bimolecular fluorescence complementation, Förster resonance energy transfer, and coimmunoprecipitation approaches revealed that SERK1 interacted biochemically with EMS1. Transphosphorylation of EMS1 by SERK1 enhances EMS1 kinase activity. Among 12 in vitro autophosphorylation and transphosphorylation sites identified by tandem mass spectrometry, seven of them were found to be critical for EMS1 autophosphorylation activity. Furthermore, complementation test results suggest that phosphorylation of EMS1 is required for its function in anther development. Collectively, these data provide genetic and biochemical evidence of the interaction and phosphorylation between SERK1/2 and EMS1 in anther development., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

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., (© 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

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.

It has long been argued that light from a laser diode is superior to light from a light-emitting diode (LED) in terms of its effect on biological tissues. In order to shed light on this ongoing debate, we compared the antimicrobial effect of light emitted from a 405-nm LED with that of a 405-nm laser on methicillin-resistant Staphylococcus aureus (MRSA) at comparable fluences. We cultured 5 × 10(6) CFU/ml MRSA on tryptic soy agar and then irradiated culture plates once, twice, or thrice with either LED or laser light using 40, 54, 81, or 121 J/cm(2) fluence at 15-, 30-, or 240-min time interval between irradiation. Cultures were incubated immediately after irradiation at 37 °C for 24 h before imaging and counting remnant bacterial colonies. Regardless of the device used, LED or laser, irradiation at each fluence resulted in statistically significant bacterial growth suppression compared to non-irradiated controls (p < 0.0001). The antimicrobial effect of both light sources, LED and laser, was not statistically different at each fluence in 35 of the 36 experimental trials. Bacterial growth suppression achieved with either source of light increased with repeated irradiation, particularly at the 15- or 30-min treatment time interval. Thus, we conclude that the antimicrobial effect of 405-nm laser and 405-nm LED on MRSA is similar; neither has a superior antimicrobial effect when compared to the other.

Dickeya dadantii is a globally dispersed phytopathogen which causes diseases on a wide range of host plants. This pathogen utilizes the type III secretion system (T3SS) to suppress host defense responses, and secretes pectate lyase (Pel) to degrade the plant cell wall. Although the regulatory small RNA (sRNA) RsmB, cyclic diguanylate monophosphate (c-di-GMP) and flagellar regulator have been reported to affect the regulation of these two virulence factors or multiple cell behaviours such as motility and biofilm formation, the linkage between these regulatory components that coordinate the cell behaviours remain unclear. Here, we revealed a sophisticated regulatory network that connects the sRNA, c-di-GMP signalling and flagellar master regulator FlhDC. We propose multi-tiered regulatory mechanisms that link the FlhDC to the T3SS through three distinct pathways including the FlhDC-FliA-YcgR3937 pathway; the FlhDC-EcpC-RpoN-HrpL pathway; and the FlhDC-rsmB-RsmA-HrpL pathway. Among these, EcpC is the most dominant factor for FlhDC to positively regulate T3SS expression., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Förster resonance energy transfer (FRET) is a nonradiative process for the transfer of energy from an optically excited donor molecule (D) to an acceptor molecule (A) in the ground state. The underlying theory predicting the dependence of the FRET efficiency on the sixth power of the distance between D and A has stood the test of time. In contrast, a comprehensive kinetic-based theory developed recently for FRET efficiencies among multiple donors and acceptors in multimeric arrays has waited for further testing. That theory has been tested in the work described in this article using linked fluorescent proteins located in the cytoplasm and at the plasma membrane of living cells. The cytoplasmic constructs were fused combinations of Cerulean as donor (D), Venus as acceptor (A), and a photo-insensitive molecule (Amber) as a nonfluorescent (N) place holder: namely, NDAN, NDNA, and ADNN duplexes, and the fully fluorescent quadruplex ADAA. The membrane-bound constructs were fused combinations of GFP2 as donor (D) and eYFP as acceptor (A): namely, two fluorescent duplexes (i.e., DA and AD) and a fluorescent triplex (ADA). According to the theory, the FRET efficiency of a multiplex such as ADAA or ADA can be predicted from that of analogs containing a single acceptor (e.g., NDAN, NDNA, and ADNN, or DA and AD, respectively). Relatively small but statistically significant differences were observed between the measured and predicted FRET efficiencies of the two multiplexes. While elucidation of the cause of this mismatch could be a worthy endeavor, the discrepancy does not appear to question the theoretical underpinnings of a large family of FRET-based methods for determining the stoichiometry and quaternary structure of complexes of macromolecules in living cells., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The sigma-1 receptor (S1R) is a 223-amino-acid membrane protein that resides in the endoplasmic reticulum and the plasma membrane of some mammalian cells. The S1R is regulated by various synthetic molecules including (+)-pentazocine, cocaine and haloperidol and endogenous molecules such as sphingosine, dimethyltryptamine and dehydroepiandrosterone. Ligand-regulated protein chaperone functions linked to oxidative stress and neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and neuropathic pain have been attributed to the S1R. Several client proteins that interact with S1R have been identified including various types of ion channels and G-protein coupled receptors (GPCRs). When S1R constructs containing C-terminal monomeric GFP2 and YFP fusions were co-expressed in COS-7 cells and subjected to FRET spectrometry analysis, monomers, dimers and higher oligomeric forms of S1R were identified under non-liganded conditions. In the presence of the prototypic S1R agonist, (+)-pentazocine, however, monomers and dimers were the prevailing forms of S1R. The prototypic antagonist, haloperidol, on the other hand, favoured higher order S1R oligomers. These data, in sum, indicate that heterologously expressed S1Rs occur in vivo in COS-7 cells in multiple oligomeric forms and that S1R ligands alter these oligomeric structures. We suggest that the S1R oligomerization states may regulate its function(s).

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.

Förster resonance energy transfer (FRET) is an exquisitely sensitive method for detection of molecular interactions and conformational changes in living cells. The recent advent of fluorescence imaging technology with single-molecule (or molecular-complex) sensitivity, together with refinements in the kinetic theory of FRET, provide the necessary tool kits for determining the stoichiometry and relative disposition of the protomers within protein complexes (i.e., quaternary structure) of membrane receptors and transporters in living cells. In contrast to standard average-based methods, this method relies on the analysis of distributions of apparent FRET efficiencies, E(app), across the image pixels of individual cells expressing proteins of interest. The most probable quaternary structure of the complex is identified from the number of peaks in the E(app) distribution and their dependence on a single parameter, termed pairwise FRET efficiency. Such peaks collectively create a unique FRET spectrum corresponding to each oligomeric configuration of the protein. Therefore, FRET could quite literally become a spectrometric method--akin to that of mass spectrometry--for sorting protein complexes according to their size and shape., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

The endoplasmic reticulum transmembrane receptor Ire1 senses over-accumulation of unfolded proteins in the endoplasmic reticulum and initiates the unfolded protein response (UPR). The cytoplasmic portion of Ire1 has a protein kinase domain (KD) and a kinase extension nuclease (KEN) domain that cleaves an mRNA for encoding the Hac1 transcription factor needed to express UPR genes. During this UPR signaling, Ire1 proteins self-assemble into an oligomer of dimers, which essentially requires autophosphorylation of a constituent activation loop in the KD. However, it is not clear how dimerization, autophosphorylation, and KEN domain function are precisely coordinated. In this study, we uncoupled the KD and KEN domain functions, by removing the activation loop along with an extended region that we called the auto-inhibitory region (AIR), or by swapping the activation loop with a homologous loop from phosphorylase kinase 1 (Ire1(PHK)). Both Ire1(ΔAIR) and Ire1(PHK) activated the UPR even when either protein contained a mutation (D797A) that abolished the ability of Ire1 KD to transfer phosphates to the AIR. Neither protein functioned when containing mutations in key ATP binding residues (E746A and N749A) or in residues that disrupted Ire1 dimer interface (W426A or R697D). We interpret these results as evidence supporting the notion that the primary function of the kinase domain is to autophosphorylate the AIR in order to relieve auto-inhibition and that ADP acts as a switch to activate the KEN domain-catalyzed HAC1 mRNA cleavage., (Published by Elsevier Ltd.)

The literature on GPCR (G-protein-coupled receptor) homo-oligomerization encompasses conflicting views that range from interpretations that GPCRs must be monomeric, through comparatively newer proposals that they exist as dimers or higher-order oligomers, to suggestions that such quaternary structures are rather ephemeral or merely accidental and may serve no functional purpose. In the present study we use a novel method of FRET (Förster resonance energy transfer) spectrometry and controlled expression of energy donor-tagged species to show that M(3)Rs (muscarinic M(3) acetylcholine receptors) at the plasma membrane exist as stable dimeric complexes, a large fraction of which interact dynamically to form tetramers without the presence of trimers, pentamers, hexamers etc. That M(3)R dimeric units interact dynamically was also supported by co-immunoprecipitation of receptors synthesized at distinct times. On the basis of all these findings, we propose a conceptual framework that may reconcile the conflicting views on the quaternary structure of GPCRs.

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 Förster 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.

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. Förster (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.

Dielectric spectroscopy is a powerful tool for characterizing and classifying materials based on their electrical properties. In order to perform dielectric measurements on a sample with spatially varying properties, the measuring probe typically is repositioned manually on the surface of the sample for each measurement. In this paper, we present a novel technique, based on a reconfigurable multielectrode array, which facilitates the recording of measurements at various different spatial locations without physically moving the measuring electrodes. By electronically selecting one of the electrodes as the inner line and connecting the remainder of the electrodes together to form the outer line, an open-ended coaxial probe is created, which can be repositioned by simply selecting different electrode combinations; hence the name of a "traveling" coaxial probe. The geometric factor, or the cell constant, of each coaxial probe in the array was estimated from measurements on saline solutions with known electrical characteristics. In order to validate the setup for measurement of dielectric properties of biological cells, the plasma membrane capacitance and cytoplasm conductivity of yeast cells suspended in aqueous solutions were measured and compared to results from published reports. Dielectric spectroscopy imaging was carried out on tissue phantoms made of an agar gel with inclusions consisting of concentrated yeast cell suspensions. Measurements were performed on the phantoms, and the dielectric data were spatially mapped with respect to electrode location. The spatial electrical data correlated precisely with locations of yeast cell inclusions within the phantoms.

Fluorescence resonance energy transfer (FRET), measured by fluorescence intensity-based microscopy and fluorescence lifetime imaging, has been used to estimate the size of oligomers formed by the M(2) muscarinic cholinergic receptor. The approach is based on the relationship between the apparent FRET efficiency within an oligomer of specified size (n) and the pairwise FRET efficiency between a single donor and a single acceptor (E). The M(2) receptor was fused at the N terminus to enhanced green or yellow fluorescent protein and expressed in Chinese hamster ovary cells. Emission spectra were analyzed by spectral deconvolution, and apparent efficiencies were estimated by donor-dequenching and acceptor-sensitized emission at different ratios of enhanced yellow fluorescent protein-M(2) receptor to enhanced green fluorescent protein-M(2) receptor. The data were interpreted in terms of a model that considers all combinations of donor and acceptor within a specified oligomer to obtain fitted values of E as follows: n = 2, 0.495 +/- 0.019; n = 4, 0.202 +/- 0.010; n = 6, 0.128 +/- 0.006; n = 8, 0.093 +/- 0.005. The pairwise FRET efficiency determined independently by fluorescence lifetime imaging was 0.20-0.24, identifying the M(2) receptor as a tetramer. The strategy described here yields an explicit estimate of oligomeric size on the basis of fluorescence properties alone. Its broader application could resolve the general question of whether G protein-coupled receptors exist as dimers or larger oligomers. The size of an oligomer has functional implications, and such information can be expected to contribute to an understanding of the signaling process.

Current methods for analysis of data from studies of protein-protein interactions using fluorescence resonance energy transfer (FRET) emerged from several decades of research using wide-field microscopes and spectrofluorometers to measure fluorescence from individual cells or cell populations. Inherent to most measurements is an averaging of the distributions of FRET efficiencies over large populations of protein complexes, which washes out information regarding the stoichiometry and structure of protein complexes. Although the introduction of laser-scanning microscopes in principle could facilitate quantification of the distributions of FRET efficiencies in live cells, only comparatively recently did this potential fully materialize, through development of spectral- or lifetime-based approaches. To exploit this new opportunity in molecular imaging, it is necessary to further develop theoretical models and methods of data analysis. Using Monte Carlo simulations, we investigated FRET in homogenous and inhomogeneous spatial distributions of molecules. Our results indicate that an analysis based on distributions of FRET efficiencies presents significant advantages over the average-based approach, which include allowing for proper identification of biologically relevant FRET. This study provides insights into the effect of molecular crowding on FRET, and it offers a basis for information extraction from distributions of FRET efficiencies using simulations-based data fitting., (Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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.

Various tissue properties have been used in the past and present as metrics which can serve to discriminate healthy from diseased tissue. Electromagnetic absorption (of x-rays and optical signals), scattering of near-infrared light, and electrical impedance are a few such parameters. In order to serve as discriminants for diseased (e.g., neoplastic) tissue, the characteristics of these tissues must first be precisely determined. In this paper, we consider the electrical impedance properties of tissues and cell aggregates, and present the design of a reconfigurable electrode array which is capable of providing a well-defined electromagnetic interface to the tissue under study, for characterization in the 0.01-30 MHz range. The configuration of array elements may be easily changed under digital control, allowing for various electromagnetic field configurations to be applied to the tissue under study. The array is designed to interface to four-point as well as two-point impedance instrumentation, and may be used for two-dimensional bioimaging systems based on electrical impedances. The design may be scaled to higher frequencies and smaller dimensions, allowing for studies of electrical properties at the cellular level.

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.

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-alpha 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.

A theoretical model is proposed for the apparent efficiency of fluorescence (Förster) resonance energy transfer (FRET) in mixtures of free monomers and homo-oligomeric protein complexes of uniform size. The model takes into account possible pathways for transfer of optical excitations from single donors to multiple acceptors and from multiple donors (non-simultaneously) to single acceptors. This necessary departure from the standard theory has been suggested in the literature, but it has only been successfully implemented for a few particular cases, such as for particular geometries of the oligomers. The predictions of the present theoretical model differ significantly from those of the standard theory, with the exception of the case of dimers, for which agreement is observed. This model therefore provides new insights into the FRET behavior of oligomers comprising more than two monomers, and also suggests means for determining the size of oligomeric protein complexes as well as the proportion of associated and unassociated monomers.