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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.)