Resonance Energy Transfer Microscopy Research Articles

Studying Nuclear Receptor Complexes in the Cellular Environment.

The ligand-regulated structure and biochemistry of nuclear receptor complexes are commonly determined by in vitro studies of isolated receptors, cofactors, and their fragments. However, in the living cell, the complexes that form are governed not just by the relative affinities of isolated cofactors for the receptor but also by the cell-specific sequestration or concentration of subsets of competing or cooperating cofactors, receptors, and other effectors into distinct subcellular domains and/or their temporary diversion into other cellular activities. Most methods developed to understand nuclear receptor function in the cellular environment involve the direct tagging of the nuclear receptor or its cofactors with fluorescent proteins (FPs) and the tracking of those FP-tagged factors by fluorescence microscopy. One of those approaches, Förster resonance energy transfer (FRET) microscopy, quantifies the transfer of energy from a higher energy "donor" FP to a lower energy "acceptor" FP attached to a single protein or to interacting proteins. The amount of FRET is influenced by the ligand-induced changes in the proximities and orientations of the FPs within the tagged nuclear receptor complexes, which is an indicator of the structure of the complexes, and by the kinetics of the interaction between FP-tagged factors. Here, we provide a guide for parsing information about the structure and biochemistry of nuclear receptor complexes from FRET measurements in living cells.

Fluorescence Resonance Energy Transfer Microscopy for Measuring Chromatin Complex Structure and Dynamics.

The Polycomb group (PcG) proteins form regulatory complexes that modify the chromatin structure and silence their target genes. Recent works have found that the composition of Polycomb complexes is highly dynamic. Defining the different protein components of each complex is fundamental for better understanding their biological functions. Fluorescent resonance energy transfer (FRET) is a powerful tool to measure protein-protein interactions, in nanometer order and in their native cellular environment. Here we describe the preparation and execution of a typical FRET experiment using CFP-tagged protein as donor and YFP-tagged protein as acceptor. We further show that FRET can be used in a competition assay to measure binding affinities of different components of the same chromatin complex.

Sequence-function correlations and dynamics of ERG isoforms. ERG8 is the black sheep of the family

The transcription factor ERG is known to have divergent roles. On one hand, it acts as differentiation factor of endothelial cells. On the other hand, it has pathological roles in various cancers. Genomic analyses of the ERG gene show that it gives rise to several isoforms. However, functional differences between these isoforms, representing potential reasons for distinct effects in diverse cell types have not been addressed in detail so far. We set out to investigate the major protein isoforms and found that ERG8 contains a unique C-terminus. This isoform, when expressed as GFP-fusion protein, localized mainly to the cytosol, whereas the other major isoforms (ERG1-4) were predominantly nuclear. Using site directed mutagenesis and laser scanning microscopy of live cells, we could identify nuclear localization (NLS) and nuclear export sequences (NES). These analyses indicated that ERG8 lacks a classical NLS and the DNA-binding domain, but holds an additional NES within its distinctive C-terminus. All the tested isoforms were shuttling between nucleus and cytosol and showed a high degree of mobility. ERG's 1 to 4 were transcriptionally active on ERG-promoter elements whereas ERG8 was inactive, which is in line with the absence of a DNA-binding domain. Fluorescence resonance energy transfer (FRET) microscopy revealed that ERG8 can bind to the transcriptionally active ERG's. Knockdown of ERG8 in endothelial cells resulted in upregulation of endogenous ERG-transcriptional activity implying ERG8 as an inhibitor of the active ERG isoforms. Quantitative PCR revealed a different ratio of active ERG's to ERG8 in cancer- versus non-transformed cells.

Cdc42 and RhoA reveal different spatio-temporal dynamics upon local stimulation with Semaphorin-3A.

Small RhoGTPases, such as Cdc42 and RhoA, are key players in integrating external cues and intracellular signaling pathways that regulate growth cone (GC) motility. Indeed, Cdc42 is involved in actin polymerization and filopodia formation, whereas RhoA induces GC collapse and neurite retraction through actomyosin contraction. In this study we employed Förster Resonance Energy Transfer (FRET) microscopy to study the spatio-temporal dynamics of Cdc42 and RhoA in GCs in response to local Semaphorin-3A (Sema3A) stimulation obtained with lipid vesicles filled with Sema3A and positioned near the selected GC using optical tweezers. We found that Cdc42 and RhoA were activated at the leading edge of NG108-15 neuroblastoma cells during spontaneous cycles of protrusion and retraction, respectively. The release of Sema3A brought to a progressive activation of RhoA within 30 s from the stimulus in the central region of the GC that collapsed and retracted. In contrast, the same stimulation evoked waves of Cdc42 activation propagating away from the stimulated region. A more localized stimulation obtained with Sema3A coated beads placed on the GC, led to Cdc42 active waves that propagated in a retrograde manner with a mean period of 70 s, and followed by GC retraction. Therefore, Sema3A activates both Cdc42 and RhoA with a complex and different spatial-temporal dynamics.

Spectral wide-field microscopic fluorescence resonance energy transfer imaging in live cells.

With its precise, sensitive, and nondestructive features, spectral unmixing-based fluorescence resonance energy transfer (FRET) microscopy has been widely applied to visualize intracellular biological events. In this report, we set up a spectral wide-field microscopic FRET imaging system by integrating a varispec liquid crystal tunable filter into a wide-field microscope for quantitative FRET measurement in living cells. We implemented a representative emission-spectral unmixing-based FRET measurement method on this platform to simultaneously acquire pixel-to-pixel images of both FRET efficiency (E ) and acceptor-to-donor concentration ratio (R C ) in living HepG2 cells expressing fusion proteins in the presence or absence of free donors and acceptors and obtained consistent results with other instruments and methods. This stable and low-cost spectral wide-field microscopic FRET imaging system provides a new toolbox for imaging molecular events with high spatial resolution in living cells.

Angiotensin II stimulates internalization and degradation of arterial myocyte plasma membrane BK channels to induce vasoconstriction.

Arterial smooth muscle cells (myocytes) express large-conductance Ca(2+)-activated K(+) (BK) channel α and auxiliary β1 subunits that modulate arterial contractility. In arterial myocytes, β1 subunits are stored within highly mobile rab11A-positive recycling endosomes. In contrast, BKα subunits are primarily plasma membrane-localized. Trafficking pathways for BKα and whether physiological stimuli that regulate arterial contractility alter BKα localization in arterial myocytes are unclear. Here, using biotinylation, immunofluorescence resonance energy transfer (immunoFRET) microscopy, and RNAi-mediated knockdown, we demonstrate that rab4A-positive early endosomes traffic BKα to the plasma membrane in myocytes of resistance-size cerebral arteries. Angiotensin II (ANG II), a vasoconstrictor, reduced both surface and total BKα, an effect blocked by bisindolylmaleimide-II, concanavalin A, and dynasore, protein kinase C (PKC), internalization, and endocytosis inhibitors, respectively. In contrast, ANG II did not reduce BKα mRNA, and sodium nitroprusside, a nitric oxide donor, did not alter surface BKα protein over the same time course. MG132 and bafilomycin A, proteasomal and lysosomal inhibitors, respectively, also inhibited the ANG II-induced reduction in surface and total BKα, resulting in intracellular BKα accumulation. ANG II-mediated BK channel degradation reduced BK currents in isolated myocytes and functional responses to iberiotoxin, a BK channel blocker, and NS1619, a BK activator, in pressurized (60 mmHg) cerebral arteries. These data indicate that rab4A-positive early endosomes traffic BKα to the plasma membrane in arterial myocytes. We also show that ANG II stimulates PKC-dependent BKα internalization and degradation. These data describe a unique mechanism by which ANG II inhibits arterial myocyte BK currents, by reducing surface channel number, to induce vasoconstriction.

Image‐based systems biology

Image‐based systems biology

FRAP, FLIM, and FRET: Detection and analysis of cellular dynamics on a molecular scale using fluorescence microscopy.

The combination of fluorescent-probe technology plus modern optical microscopes allows investigators to monitor dynamic events in living cells with exquisite temporal and spatial resolution. Fluorescence recovery after photobleaching (FRAP), for example, has long been used to monitor molecular dynamics both within cells and on cellular surfaces. Although bound by the diffraction limit imposed on all optical microscopes, the combination of digital cameras and the application of fluorescence intensity information on large-pixel arrays have allowed such dynamic information to be monitored and quantified. Fluorescence lifetime imaging microscopy (FLIM), on the other hand, utilizes the information from an ensemble of fluorophores to probe changes in the local environment. Using either fluorescence-intensity or lifetime approaches, fluorescence resonance energy transfer (FRET) microscopy provides information about molecular interactions, with Ångstrom resolution. In this review, we summarize the theoretical framework underlying these methods and illustrate their utility in addressing important problems in reproductive and developmental systems.

Visualization of the dynamics of PSD-95 and Kir2.1 interaction by fluorescence lifetime-based resonance energy transfer imaging

Summary Many cellular processes are orchestrated by protein–protein interactions that allow the formation of protein networks involved in subcellular compartmentalization, communication, and signalling. Postsynaptic density protein 95 (PSD-95), a member of the membrane-associated guanylate kinase protein (MAGuK) family, is a central scaffold protein of the postsynaptic density (PSD) of excitatory synapses in the mammalian central nervous system. PSD-95 serves as a matrix for targeting and accumulation of yet other PSD proteins including ion channels and receptors via its three N-terminal PDZ (PSD-95, discs large, zonula occludens-1) domains. However, the stoichiometry of PSD-95 and its binding partners in such complexes and the dynamic regulation of their interactions remain elusive. Here, we have investigated the protein–protein interaction between PSD-95 and the inward rectifier potassium channel Kir2.1, which we consider as a model for other PDZ domain based interactions at synapses. By using Forster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM), we show that PSD-95 and Kir2.1 directly interact within clusters which are formed at the plasma membrane of culture cells. Our in vivo FRET data indicate that Kir2.1 binds to more than one PDZ domain of PSD-95, suggesting a structural model of synergistic target binding by the first two PDZ domains of the scaffold protein. We show that the cluster formation is induced by the channel whereas PSD-95 alone does not form clusters. The interaction of PSD-95 and Kir2.1 is dynamically regulated by protein kinase A (PKA) mediated phosphorylation, which is directly visualized and quantified in living cells and in real time.

Three-color confocal Förster (or fluorescence) resonance energy transfer microscopy: Quantitative analysis of protein interactions in the nucleation of actin filaments in live cells.

Experiments using live cell 3-color Förster (or fluorescence) resonance energy transfer (FRET) microscopy and corresponding in vitro biochemical reconstitution of the same proteins were conducted to evaluate actin filament nucleation. A novel application of 3-color FRET data is demonstrated, extending the analysis beyond the customary energy-transfer efficiency (E%) calculations. MDCK cells were transfected for coexpression of Teal-N-WASP/Venus-IQGAP1/mRFP1-Rac1, Teal-N-WASP/Venus-IQGAP1/mRFP1-Cdc42, CFP-Rac1/Venus-IQGAP1/mCherry-actin, or CFP-Cdc42/Venus-IQGAP1/mCherry-actin, and with single-label equivalents for spectral bleedthrough correction. Using confirmed E% as an entry point, fluorescence levels and related ratios were correlated at discrete accumulating levels at cell peripheries. Rising ratios of CFP-Rac1:Venus-IQGAP1 were correlated with lower overall actin fluorescence, whereas the CFP-Cdc42:Venus-IQGAP1 ratio correlated with increased actin fluorescence at low ratios, but was neutral at higher ratios. The new FRET analyses also indicated that rising levels of mRFP1-Cdc42 or mRFP1-Rac1, respectively, promoted or suppressed the association of Teal-N-WASP with Venus-IQGAP1. These 3-color FRET assays further support our in vitro results about the role of IQGAP1, Rac1, and Cdc42 in actin nucleation, and the differential impact of Rac1 and Cdc42 on the association of N-WASP with IQGAP1. In addition, this study emphasizes the power of 3-color FRET as a systems biology strategy for simultaneous evaluation of multiple interacting proteins in individual live cells.

Cytoplasmic delivery and selective, multicomponent labeling with oligoarginine-linked protein tags.

Strategies that leverage bio-orthogonal interactions between small molecule ligands and genetically encoded amino acid sequences can be used to attach high-performance fluorophores to proteins in living cells. However, a major limitation of chemical protein labeling is that cells' plasma membranes are impermeable to many useful probes and biolabels. Here, we show that conjugation to nonaarginine, a cell penetrating peptide (CPP), enables passive cytoplasmic delivery of otherwise membrane-impermeant, small molecule protein labels. Heterodimers consisting of a luminescent Tb(3+) complex, Lumi4, linked to benzyl guanine, benzyl cytosine, and trimethoprim were conjugated to the peptide CysArg9 with a reducible disulfide linker. When added to culture medium, the peptide conjugates rapidly (<30 min) enter the cytoplasm and diffuse freely throughout cells. The benzyl guanine, benzyl cytosine, and trimethoprim derivatives bind selectively to fusion proteins tagged with SNAP-Tag, CLIP-Tag, and Escherichia coli dihydrofolate reductase (eDHFR), respectively. Furthermore, eDHFR and SNAP-Tag fusions can be labeled with Lumi4 analogues in the same cell, and this labeling can be detected using two-color, time-gated Förster resonance energy transfer (FRET) microscopy. Finally, we present quantitative data showing that cytoplasmic uptake of nonaarginine-conjugated probes occurs in multiple cell types (MDCK, HeLa, NIH 3T3), most cells in a culture (>75%) are loaded with probe, and the cellular probe concentration can be controlled by varying incubation conditions. CPP-mediated delivery of Lumi4-linked protein labels will greatly increase the utility of lanthanide-based FRET microscopy. Moreover, our results strongly suggest that this approach can be adapted to deliver a wide variety of protein-targeted fluorophores or other functional probes that were previously unavailable for intracellular imaging studies.

Multicolor time-resolved Förster resonance energy transfer microscopy reveals the impact of GPCR oligomerization on internalization processes.

Identifying the interacting partners and the dynamics of the molecular networks constitutes the key point in understanding cellular processes. Different methods often based on energy transfer strategies have been developed to examine the molecular dynamics of protein complexes. However, these methods suffer a couple of drawbacks: a single complex can be studied at a time, and its localization and tracking cannot generally be investigated. Here, we report a multicolor time-resolved Förster resonance energy transfer microscopy method that allows the identification of up to 3 different complexes simultaneously, their localization in cells, and their tracking after activation. Using this technique, we studied GPCR oligomerization and internalization in human embryonic kidney 293 cells. We definitively show that receptors can internalize as oligomers and that receptor coexpression deeply impacts oligomer internalization processes.

Stepping between Membrane Microdomains

Lateral membrane microdomains are postulated to be involved in a number of important functions in mammalian cells, such as membrane trafficking, exocytosis, endocytosis, signal transduction, and protein activity (1,2). These putative cell microdomains have been the subject of extensive research using a variety of biophysical and biochemical techniques, including detergent extraction (2), fluorescence microscopy (3), electron microscopy (4), fluorescence resonance energy transfer microscopy (5), fluorescence correlation spectroscopy (6), fluorescence recovery after photobleaching (7), and single-molecule tracking (8).

Parkin maintains mitochondrial levels of the protective Parkinson's disease-related enzyme 17-β hydroxysteroid dehydrogenase type 10.

Mutations of the PARK2 and PINK1 genes, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial serine/threonine kinase PINK1, respectively, cause autosomal recessive early-onset Parkinson's disease (PD). Parkin and PINK1 cooperate in a biochemical mitochondrial quality control pathway regulating mitochondrial morphology, dynamics and clearance. This study identifies the multifunctional PD-related mitochondrial matrix enzyme 17-β hydroxysteroid dehydrogenase type 10 (HSD17B10) as a new Parkin substrate. Parkin overproduction in cells increased mitochondrial HSD17B10 abundance by a mechanism involving ubiquitin chain extension, whereas PARK2 downregulation or deficiency caused mitochondrial HSD17B10 depletion in cells and mice. HSD17B10 levels were also found to be low in the brains of PD patients with PARK2 mutations. Confocal and Förster resonance energy transfer (FRET) microscopy revealed that HSD17B10 recruited Parkin to the translocase of the outer membrane (TOM), close to PINK1, both in functional mitochondria and after the collapse of mitochondrial membrane potential (ΔΨm). PD-causing PARK2 mutations impaired interaction with HSD17B10 and the HSD17B10-dependent mitochondrial translocation of Parkin. HSD17B10 overproduction promoted mitochondrial elongation and mitigated CCCP-induced mitochondrial degradation independently of enzymatic activity. These effects were abolished by overproduction of the fission-promiting dynamin-related protein 1 (Drp1). By contrast, siRNA-mediated HSD17B10 silencing enhanced mitochondrial fission and mitophagy. These findings suggest that the maintenance of appropriate mitochondrial HSD17B10 levels is one of the mechanisms by which Parkin preserves mitochondrial quality. The loss of this protective mechanism may contribute to mitochondrial dysfunction and neuronal degeneration in autosomal recessive PD.

Single-Molecule Pull-Down FRET to Dissect the Mechanisms of Biomolecular Machines.

Spliceosomes are multimegadalton RNA-protein complexes responsible for the faithful removal of noncoding segments (introns) from pre-messenger RNAs (pre-mRNAs), a process critical for the maturation of eukaryotic mRNAs for subsequent translation by the ribosome. Both the spliceosome and ribosome, as well as many other RNA and DNA processing machineries, contain central RNA components that endow biomolecular complexes with precise, sequence-specific nucleic acid recognition, and versatile structural dynamics. Single-molecule fluorescence (or Förster) resonance energy transfer (smFRET) microscopy is a powerful tool for the study of local and global conformational changes of both simple and complex biomolecular systems involving RNA. The integration of biochemical tools such as immunoprecipitation with advanced methods in smFRET microscopy and data analysis has opened up entirely new avenues toward studying the mechanisms of biomolecular machines isolated directly from complex biological specimens, such as cell extracts. Here, we detail the general steps for using prism-based total internal reflection fluorescence microscopy in exemplary single-molecule pull-down FRET studies of the yeast spliceosome and discuss the broad application potential of this technique.

Raising the Speed Limit on 3D-3Way FRET Microscopy

The spatial organization of protein-protein interactions inside of cells is an important component of cellular signaling. These interactions can be imaged using fluorescent resonance energy transfer (FRET) microscopy methods. We have described a generalized mathematical solution and method for any number of interacting FRET pairs (N-Way FRET). Here, we combine N-Way FRET with our three-dimensional FRET stoichiometry reconstruction (3D-FSR) to allow 3D-3Way-FRET image deconvolution. By developing a multi-camera instrument, we are able to acquire the 6 images needed per z-plane with sub-second time resolution, allowing observation of live cell dynamics. Furthermore, we implemented improved fast Fourier transforms and parallelized calculations to achieve a 10-fold decrease in the computational time required to reconstruct 3D-3Way-FRET data. This reduction has made it feasible to analyze dynamic 2Way and 3Way FRET data with 3D reconstructions, increase the number of reconstruction iterations, as well as explore stopping criteria for the algorithm.

Single Molecule FRET Analysis of the Closed and Open States of a DNA Origami Box

DNA origami is an excellent tool for building artificial nanostructures using only DNA as the building block. In this technique, a long circular single stranded scaffold is molded into a predesigned shape by short single stranded staple strands, and many different structures can be produced including 2- and 3-dimensional structures which can be both static and dynamic. We designed a DNA box 43x36x36 nm3 in size that can be opened in a controlled manner. Two DNA locks hold the box closed, which can be opened as a response to sensing of specific DNA sequences. The closed and open states of the box are here investigated by single molecule Förster resonance energy transfer (FRET) microscopy. We find that the measured FRET efficiency for a closed box depends on the buffer ion concentration, which can affect the DNA origami structure. Single-molecule FRET measurements of surface immobilized DNA boxes offer the possibility to investigate a wide range of ion concentration and provide local structural insights close to the fluorophores position.

Measurement of ROS Levels and Membrane Potential Dynamics in the Intact Carotid Body Ex Vivo.

Reactive oxygen species (ROS) generated by the NADPH oxidase have been proposed to play an important role in the carotid body (CB) oxygen sensing process (Cross et al. 1990). Up to now it remains unclear whether hypoxia causes an increase or decrease of CB ROS levels. We transfected CBs with the ROS sensitive HSP-FRET construct and subsequently measured the intracellular redox state by means of Förster resonance energy transfer (FRET) microscopy. In a previous study we found both increasing and decreasing ROS levels under hypoxic conditions. The transition from decreasing to increasing ROS levels coincided with the change of the caging system from ambient environment caging (AEC) to individually ventilated caging (IVC) (Bernardini A, Brockmeier U, Metzen E, Berchner-Pfannschmidt U, Harde E, Acker-Palmer A, Papkovsky D, Acker H, Fandrey J, Type I cell ROS kinetics under hypoxia in the intact mouse carotid body ex vivo: a FRET based study. Am J Physiol Cell Physiol. doi: 10.1152/ajpcell.00370.2013 , 2014). In this work we analyze hypoxia induced ROS reaction of animals from an IVC system that had been exposed to AEC conditions for 5 days. The results further support the hypothesis of an important impact of the caging system on CB ROS reaction.

Quantitative single molecule FRET efficiencies using TIRF microscopy.

Förster resonance energy transfer (FRET) microscopy at the single molecule level has the potential to yield information on intra and intermolecular distances within the 2-10 nm range of molecules or molecular complexes that undergo frequent conformation changes. A pre-requirement for obtaining accurate distance information is to determine quantitative instrument independent FRET efficiency values. Here, we applied and evaluated a procedure to determine quantitative FRET efficiencies directly from individual fluorescence time traces of surface immobilized DNA molecules without the need for external calibrants. To probe the robustness of the approach over a wide range of FRET efficiencies we used a set of doubly labelled double stranded DNA samples, where the acceptor position was varied systematically. Interestingly, we found that fluorescence contributions arising from direct acceptor excitation following donor excitation are intrinsically taken into account in these conditions as other correction factors can compensate for inaccurate values of these parameters. We give here guidelines, that can be used through tools within the iSMS software (), for determining quantitative FRET and assess uncertainties linked with the procedure. Our results provide insights into the experimental parameters governing quantitative FRET determination, which is essential for obtaining accurate structural information from a wide range of biomolecules.

Ligand-engaged TCR is triggered by Lck not associated with CD8 coreceptor.

The earliest molecular events in T cell recognition have not yet been fully described, and the initial T cell receptor (TCR) triggering mechanism remains a subject of controversy. Here, using TIRF/FRET microscopy, we observe a two-stage interaction between TCR, CD8, and MHCp. There is an early (within seconds) interaction between CD3ζ and the coreceptor CD8 that is independent of the binding of CD8 to MHC, but that requires CD8 association with Lck. Later (several minutes) CD3ζ-CD8 interactions require CD8-MHC binding. Lck can be found free or bound to the coreceptor. This work indicates that the initial TCR triggering event is induced by free Lck.