Imaging the Environment of Green Fluorescent Protein (original) (raw)
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Insights into receptor structure and dynamics at the surface of living cells
Nature Communications, 2023
Evaluating protein structures in living cells remains a challenge. Here, we investigate Interleukin-4 receptor alpha (IL-4Rα) into which the non-canonical amino acid bicyclo[6.1.0]nonyne-lysine (BCNK) is incorporated by genetic code expansion. Bioorthogonal click labeling is performed with tetrazine-conjugated dyes. To quantify the reaction yield in situ, we develop brightness-calibrated ratiometric imaging, a protocol where fluorescent signals in confocal multicolor images are ascribed to local concentrations. Screening receptor mutants bearing BCNK in the extracellular domain uncovered site-specific variations of both click efficiency and Interleukin-4 binding affinity, indicating subtle welldefined structural perturbations. Molecular dynamics and continuum electrostatics calculations suggest solvent polarization to determine site-specific variations of BCNK reactivity. Strikingly, signatures of differential click efficiency, measured for IL-4Rα in ligand-bound and free form, mirror subangstrom deformations of the protein backbone at corresponding locations. Thus, click efficiency by itself represents a remarkably informative readout linked to protein structure and dynamics in the native plasma membrane. Site-specific manipulation of proteins by genetic code expansion (GCE), a technique that introduces non-canonical amino acids (ncAA) into the otherwise native polypeptide chain, offers fascinating possibilities for the investigation of protein function in living cells 1,2. The introduced ncAA carries bioorthogonal reactivity for the site-specific conjugation of biophysical probes featuring negligible interference with components of the cell 3,4. A variety of chemical strategies termed click labeling emerged for site-specific conjugation of sensor molecules to ncAA under physiological conditions 5. As an alternative to metal-catalyzed click reactions, inverse electron-demand Diels-Alder cycloaddition (iEDDAC) of tetrazine derivatives to ncAA side chains presenting reactive dienophiles provides superior reaction rates for fast coupling to targets expressed via GCE 6-11. Bioorthogonal click labeling with fluorescent dyes was successfully combined with biophysical single-cell methods to address common issues in receptor research, like diffusion, oligomerization states, and conformational changes 12-18. However, only few attempts have been made to systematically quantify the yields of bioorthogonal reactions per se 19. Because the experimental throughput is limited, efforts to optimize click reaction conditions can be substantial 20,21. Fluorescence fluctuation analysis provides a simple means for transforming confocal images into molecular concentration maps 22 and is therefore a powerful extension for live cell imaging 23,24. Consequently, fluorescence fluctuation analysis was introduced as a routine validation protocol in bioorthogonal labeling 25. However, even elaborate instrumentation cannot circumvent the systematic errors inherent to single-cell measurements. Cells are far-from-equilibrium systems that carry out a myriad of processes on various time scales. Data curation must be carefully executed post-measurement to extract meaningful information. To mitigate these efforts, we here develop
Fluorescent ligands, antibodies, and proteins for the study of receptors
Pharmacology & Therapeutics, 2003
Fluorescent molecules bound to receptors can show their location and, if binding is reversible, can provide pharmacological information such as affinity and proximity between interacting molecules. The spatial precision offered by visualisation transcends the diverse localisation and low molecular concentration of receptor molecules. Consequently, the relationships between receptor location and function and life cycles of receptors have become better understood as a result of fluorescent labeling. Each of these aspects contributes new insights to drug action and potential new targets. The relationships between spatial distribution of receptor and function are largely unknown. This is particularly apparent for native receptors expressed in their normal host tissues where communication between heterogeneous cell types influences receptor distribution and function. In cultured cell systems, particularly for G-protein-coupled receptors (GPCR), fluorescencebased methods have enabled the visualisation of the cycle of agonist-stimulated receptor clustering, endocytic internalisation to the perinuclear region, degradation of the receptor-ligand complex, and recycling back to the surface membrane. Using variant forms of green fluorescent protein (GFP), antibodies, or fluorescent ligands, it is possible to detect or visualise the formation of oligomeric receptor complexes. Careful selection of fluorescent molecules based on their spectral properties enables resonance energy transfer and multilabel visualisation with colocalisation studies. Fluorescent agonist and antagonist ligands are now being used in parallel with GFP to study receptor cycling in live cells. This review covers how labeling and visualisation technologies have been applied to the study of major pharmacologically important receptors and illustrates this by giving examples of recent techniques that have relied on GFP, antibodies, or fluorescent ligands alone or in combination for the purpose of studying GPCR. D
Fluorescence techniques: shedding light on ligand–receptor interactions
Trends in Pharmacological Sciences, 2000
The ability of organisms, or individual cells, to react to external chemical signals, which are detected and transduced by cell-surface receptors, is crucial for their survival. These receptors are the targets of the majority of clinically used medicines. Combinatorial genetics can provide almost unlimited numbers of mutant receptor proteins and combinatorial chemistry can produce large libraries of potential therapeutic compounds that act on these membrane receptors. What is missing for the fundamental understanding of receptor function and for the discovery of new medicines are efficient procedures to screen both ligand-receptor interactions and the subsequent functional consequences. Ultrasensitive fluorescence spectroscopic approaches, in combination with efficient labelling protocols, offer enormous possibilities for highly parallel functional bioanalytics at the micro-and nanometer level.
ChemBioChem, 2014
Classical fluorescence-based approaches to monitor ligandprotein interactions are generally hampered by the background signal of unbound ligand, which must be removed by tedious washing steps. To overcome this major limitation, we report here the first red fluorescent turn-on probes for a G protein-coupled receptor (oxytocin receptor) at the surface of living cells. The peptide ligand carbetocin was conjugated to one of the best solvatochromic (fluorogenic) dyes, Nile Red, which turns on emission when reaching the hydrophobic environment of the receptor. We showed that the incorporation of hydrophilic octa(ethylene glycol) linker between the pharmacophore and the dye minimized nonspecific interaction of the probe with serum proteins and lipid membranes, thus ensuring receptor-specific turn-on response. The new ligand was successfully applied for background-free imaging and quantification of oxytocin receptors in living cells.
Identification and super-resolution imaging of ligand-activated receptor dimers in live cells
Scientific reports, 2013
Molecular interactions are key to many chemical and biological processes like protein function. In many signaling processes they occur in sub-cellular areas displaying nanoscale organizations and involving molecular assemblies. The nanometric dimensions and the dynamic nature of the interactions make their investigations complex in live cells. While super-resolution fluorescence microscopies offer live-cell molecular imaging with sub-wavelength resolutions, they lack specificity for distinguishing interacting molecule populations. Here we combine super-resolution microscopy and single-molecule Förster Resonance Energy Transfer (FRET) to identify dimers of receptors induced by ligand binding and provide super-resolved images of their membrane distribution in live cells. By developing a two-color universal-Point-Accumulation-In-the-Nanoscale-Topography (uPAINT) method, dimers of epidermal growth factor receptors (EGFR) activated by EGF are studied at ultra-high densities, revealing pr...
Biophysics and Physicobiology, 2022
Edited by Takeharu Nagai Membrane receptors provide interfaces of various extracellular stimuli to transduce the signal into the cell. Receptors are required to possess such conflicting properties as high sensitivity and noise reduction for the cell to keep its homeostasis and appropriate responses. To understand the mechanisms by which these functions are achieved, single-molecule monitoring of the motilities of receptors and signaling molecules on the plasma membrane is one of the most direct approaches. This review article introduces several recent single-molecule imaging studies of receptors, including the author's recent work on triple-color single-molecule imaging of G protein-coupled receptors. Based on these researches, advantages and perspectives of the single-molecule imaging approach to solving the mechanisms of receptor functions are illustrated.
Critical design issues in the targeted molecular imaging of cell surface receptors
Chemical Society reviews, 2015
The imaging of cell-surface receptors can be achieved using several methods, including single photon emission tomography (SPECT) positron emission tomography (PET), optical imaging and magnetic resonance imaging (MRI). The application of targeted MRI contrast agents is particularly well-suited to this task, provided that the agents reach the desired site efficiently and selectively. In addition, they should bind reversibly to the cell-surface receptor and give rise to a large change in cellular relaxation rate, in competition with binding to the natural substrate. Such approaches offer promise in the molecular imaging of neurotransmission in the brain, using conjugates that selectively target dopamine or glutamate receptor sub-types. Strategies based on the use of competitive antagonist vectors offer particular scope, as such conjugates are generally not taken into the target cell following cell surface receptor binding, in contrast to the use of MRI contrast agents based on agonist...
Journal of Visualized Experiments, 2019
Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&B) analysis. N&B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective timeper-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.