Acceptor Photobleaching Fluorescence Resonance Energy Transfer Research Articles

Imaging-based assays for investigating functions of the RNA polymerase II elongation factor Elongin and the Elongin ubiquitin ligase.

Elongin A binds to Elongins B and C to form the RNA polymerase II transcription elongation factor Elongin. It also functions as the substrate recognition subunit of a ubiquitin ligase that is formed by binding of Elongin to Cullin protein CUL5 and RING finger protein RBX2 and that targets RNA polymerase II for ubiquitination. In this article, we describe use of acceptor photobleaching fluorescence resonance energy transfer (AP-FRET) and laser microirradiation-based assays to study regulated assembly of the Elongin ubiquitin ligase and its recruitment to regions of localized DNA damage.

γ-Secretase Modulators and Inhibitors Induce Different Conformational Changes of Presenilin 1 Revealed by FLIM and FRET.

Elucidation of γ-secretase structure and dynamic conformational changes is of importance to drug discovery targeting this enzyme. Electron microscopy analyses provided important structural information, but the dynamic changes of γ-secretase in cells need to be explored further. We found that PS1 internal fluorescence resonance energy transfer (FRET) probes can incorporate into γ-secretase complex and possess secretase activity. Our results from fluorescence lifetime image microscopy (FLIM) and acceptor photobleaching FRET show different PS1 internal FRET when PS1 fluorescent probes expressed alone or with other secretase subunits Aph1aL, Nicastrin, and Pen2, indicating that PS1 internal FRET could be applied for probing conformational change of γ-secretase complex. Further, we accessed whether γ-secretase activity interfering compounds induced different conformational changes of PS1. Our results show that both γ-secretase modulators and inhibitors affect PS1 internal FRET but in different manners. These results demonstrate that FLIM and acceptor photobleaching FRET could be applied to monitor different PS1 conformational changes in γ-secretase.

Fluorescence techniques used to measure interactions between hydroxyapatite nanoparticles and epidermal growth factor receptors.

The potential applications of nanomaterials in therapeutics are immense and to fully explore this potential, it is important to understand the interaction of nanoparticles with cellular components. To examine the interaction between nanoparticles and cell membrane receptors, this report describes the use of advanced fluorescence techniques to measure interactions between hydroxyapatite (HA) nanoparticles and epidermal growth factor receptors (EGFRs), as a model system. FITC-labelled HA nanoparticles and monomeric red fluorescent protein (mRFP)-conjugated EGFRs expressed in Chinese hamster ovary cells (CHO-K1) were generated and their interaction measured using acceptor photobleaching-fluorescence resonance energy transfer (AP-FRET) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET). Results confirmed that hydroxyapatite nanoparticles not only interacted with EGFR but also attenuated downstream EGFR signalling, possibly by hindering normal dimerization of EGFR. Furthermore, the extent of signal attenuation suggested correlation with specific surface area of the nanoparticles, whereby greater specific surface area resulted in greater downstream signal attenuation. This novel demonstration establishes fluorescence techniques as a viable method to study nanoparticle interactions with proteins such as cell surface receptors. The approach described herein can be extended to study interactions between any fluorescently labelled nanoparticle-biomolecule pair.

Optimization of FRET Microscopy for Live-Cell Imaging of Multiple Protein-Protein Interactions

Recent advances in fluorescence resonance energy transfer (FRET) microscopy have made it possible to measure simultaneously, multiple protein-protein interactions inside living cells. To further these methods, we integrated an improved multi-spectral imaging analysis method (N-way FRET) with optimized fluorophores for live-cell FRET microscopy. For 3-way FRET we determined the best cyan, yellow, and red (C-Y-R) FRET-trio by comparing the acceptor photobleaching FRET efficiencies of structurally similar linked fluorescent protein constructs. The pool of molecules examined was constrained to the available C, Y, R molecules with the best photophysical properties (CyPet/TFP1, mCitrine/YPet and TagRFP/mCherry, respectively). From this set, we determined that TFP, YPet or mCitrine and mCherry were the optimal FRET trio. For increasingly complex pathways, more fluorescent probes may be required. To extend this method to the analysis of 4-way FRET we examined fluorophore pairs with highly overlapping excitation and emission spectra (e.g. TFP and GFP or Citrine and GFP). These improvements should enhance the spatiotemporal resolution, the number of possible interacting probes and the reliability of FRET experiments leading to improved monitoring of multiple protein-protein interactions in complex pathways.

Determination of cellulase colocalization on cellulose fiber with quantitative FRET measured by acceptor photobleaching and spectrally unmixing fluorescence microscopy

The determination of cellulase distribution on the surface of cellulose fiber is an important parameter to understand when determining the interaction between cellulase and cellulose and/or the cooperation of different types of cellulases during the enzymatic hydrolysis of cellulose. In this communication, a strategy is presented to quantitatively determine the cellulase colocalization using the fluorescence resonance energy transfer (FRET) methodology, which is based on acceptor photobleaching and spectrally unmixing fluorescence microscopy. FRET monitoring of cellulase colocalization was achieved by labeling cellulases with an appropriate pair of FRET dyes and by adopting an appropriate FRET model. We describe here that the adapted acceptor photobleaching FRET method can be successfully used to quantify cellulase colocalization regarding their binding to a cellulose fiber at a resolution <10 nm. This developed quantitative FRET method is promising for further studying the interactions between cellulase and cellulose and between different types of cellulases.

Fluorescence Resonance Energy Transfer (FRET)-based Detection of Profilin–VASP Interaction

Profilins belong to a family of small G-actin binding proteins which are thought to assist in F-actin elongation at the leading edge of migrating cells through their interactions with a host of actin-binding proteins including Ena (enabled)/VASP (vasodilator stimulated phosphoprotein). Profilin's interactions with the major actin regulators have been studied almost exclusively using biochemical methods. Therefore spatiotemporal features of these protein-protein interactions have not been resolved so far. In this paper, we for the first time demonstrate the feasibility of GFP-based fluorescence resonance energy transfer (FRET) technique to detect VASP's interaction with profilin-1, a ubiquitously expressed member of profilin family of genes. Specifically, we performed acceptor photobleaching FRET in MDA-MB-231 breast cancer cells to show prominent VASP-Pfn1 interaction at the membrane ruffles near the leading edge.

S1760 The Homeodomain Transcription Factor CDx2 Regulates E Cadherin Activity By Enhancing Its Localization from the Cytoplasm to the Cell Membrane

trafficking. Methods: In the present study, we employed fluorescence resonance energy transfer (FRET) and Zeiss 510 confocal microscopy to study direct binding of NHE3 and NHERF2 in living or fixed polarized kidney epithelial cells (OK cells) that were transiently transfected with CFP-NHERF2 and YFP-NHE3. Results: Under basal conditions, NHERF2 and NHE3 exhibited 10-20% FRET signaling solely at the microvilli (an average of 10 regions of interest (ROIs) in the microvilli of each cells) but not at the juxtanuclear region. As a negative control, there was no FRET signaling with expression of YFP-GPI and CFP-NHERF2. Further, treatment with 10 μMCa2+ ionophore, which increases intracellular Ca2+, abolished the NHERF2/NHE3 FRET signaling within a minute. To further elucidate the role of Ca2+ signaling in regulation of NHE3 trafficking, we treated Caco-2/HA-NHE3 cells with carbachol (10 uM) that activates M3 muscarinic receptors and increases intracellular Ca2+ and monitored its effect on the trafficking of NHE3 via total internal reflection fluorescence (TIRF) microscopy. Carbachol reduced the surface level of NHE3 with an onset within one minute. Conclusions: 1) Under basal conditions, NHE3 binds NHERF2 in the OK cell microvilli and this binding can be quantified by acceptor photobleaching FRET. 2) Within one minute of elevation of intracellular Ca2+, the FRET signal between NHE3 and NHERF2 is abolished in OK cells. 3) Carbachol causes a similarly rapid reduction of surface NHE3 observed via TIRF. We conclude that elevation of intracellular Ca2+ leads to dissociation of NHERF2 from NHE3 at the microvillus which allows NHE3 transport activity to be inhibited by facilitating endocytosis.

AccPbFRET: An ImageJ plugin for semi-automatic, fully corrected analysis of acceptor photobleaching FRET images

BackgroundThe acceptor photobleaching fluorescence resonance energy transfer (FRET) method is widely used for monitoring molecular interactions in cells. This method of FRET, while among those with the simplest mathematics, is robust, self-controlled and independent of fluorophore amounts and ratios.ResultsAccPbFRET is a user-friendly, efficient ImageJ plugin which allows fully corrected, pixel-wise calculation and detailed, ROI (region of interest)-based analysis of FRET efficiencies in microscopic images. Furthermore, automatic registration and semi-automatic analysis of large image sets is provided, which are not available in any existing FRET evaluation software.ConclusionDespite of the widespread applicability of the acceptor photobleaching FRET technique, this is the first paper where all possible sources of major errors of the measurement and analysis are considered, and AccPbFRET is the only program which provides the complete suite of corrections – for registering image pairs, for unwanted photobleaching of the donor, for cross-talk of the acceptor and/or its photoproduct to the donor channel and for partial photobleaching of the acceptor. The program efficiently speeds up the analysis of large image sets even for novice users and is freely available.

In vivointeractions of MyoD, Id1, and E2A proteins determined by acceptor photobleaching fluorescence resonance energy transfer

MyoD, a skeletal muscle transcription factor, is rapidly degraded by the ubiquitin-proteasome system. MyoD interacts with ubiquitously expressed E2A or inhibitor of DNA binding (Id) proteins to activate or inhibit transcription, respectively. Furthermore, MyoD has been shown to modulate the ubiquitin-mediated degradation of Id1 and E2A proteins, E12 and E47. The molecular mechanisms governing these events are not clear but are hypothesized to occur via heterodimer formation. Fluorescence resonance energy transfer (FRET) is a technique for evaluation of protein-protein interactions in vivo. Using acceptor photobleaching FRET and chimeric proteins composed of MyoD, Id1, E12, E47, E12(NLS), or MyoD(NLS) and either cyan fluorescent protein or yellow fluorescent protein, we show that each of the wild-type proteins is capable of homodimerization. In addition, heterodimers form between Id1 and E2A proteins, as well as between MyoD and E2A proteins. The Id1:E2A interaction is stronger than the MyoD:E2A interaction, which is consistent with the notion that inhibition of MyoD action occurs by the sequestration of E2A proteins by Id. The stronger interaction of Id1 with E2A may also explain the decrease in the rate of ubiquitin-proteasome degradation of Id1 that is significantly greater than that of MyoD when E2A proteins are abundant. Thus, these studies extend our understanding of the molecular mechanisms of MyoD action.

Heterodimerization of y +LAT-1 and 4F2hc visualized by acceptor photobleaching FRET microscopy

y +LAT-1 and 4F2hc are the subunits of a transporter complex for cationic amino acids, located mainly in the basolateral plasma membrane of epithelial cells in the small intestine and renal tubules. Mutations in y +LAT-1 impair the transport function of this complex and cause a selective aminoaciduria, lysinuric protein intolerance (LPI, OMIM #222700), associated with severe, complex clinical symptoms. The subunits of an active transporter co-localize in the plasma membrane, but the exact process of dimerization is unclear since direct evidence for the assembly of this transporter in intact human cells has not been available. In this study, we used fluorescence resonance energy transfer (FRET) microscopy to investigate the interactions of y +LAT-1 and 4F2hc in HEK293 cells expressing y +LAT-1 and 4F2hc fused with ECFP or EYFP. FRET was quantified by measuring fluorescence intensity changes in the donor fluorophore (ECFP) after the photobleaching of the acceptor (EYFP). Increased donor fluorescence could be detected throughout the cell, from the endoplasmic reticulum and Golgi complex to the plasma membrane. Therefore, our data prove the interaction of y +LAT-1 and 4F2hc prior to the plasma membrane and thus provide evidence for 4F2hc functioning as a chaperone in assisting the transport of y +LAT-1 to the plasma membrane.

Imaging Protein Interactions by FRET Microscopy: FRET Measurements by Acceptor Photobleaching: Figure 1.

This protocol describes the detection of fluorescence resonance energy transfer (FRET) by measuring the quenching of donor emission alone. As opposed to sensitized emission measurements, photobleaching can be performed with high selectivity of the acceptor because absorption spectra are steep at their red edge, allowing the acceptor to be bleached without excitation of the donor. When using acceptor photobleaching FRET measurements, care should be taken that the photochemical product of the bleached acceptor does not have residual absorption at the donor emission and, more importantly, that it does not fluoresce in the donor spectral region. Because of mass movement of protein during the extended time required for photobleaching (typically 1-20 min), it is preferable to perform this type of FRET determination on fixed cell samples. Live-cell FRET measurements based only on donor fluorescence are more feasible using fluorescence lifetime imaging (FLIM), because lifetimes are independent of probe concentration and light path length. The former is not easy to determine in cells, and the latter means that cell shape is not a factor.

Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells

Fluorescence resonance energy transfer (FRET) refers to the nonradiative transfer of energy from one fluorescent molecule (the donor) to another fluorescent molecule (the acceptor). Measurement of FRET between two fluorophore-labeled proteins can be used to infer the subnanometer spatial and temporal characteristics of protein interactions in their native cellular environment. Multiple experimental methods exist for measuring FRET. The method that can be most widely and simply implemented, quantified, and interpreted is the acceptor-photobleaching FRET technique. In this method, the presence of FRET between a donor and acceptor is revealed upon destruction (by photobleaching) of the acceptor. Acceptor photobleaching can be exploited to detect changes in the composition and organization of subunit proteins within a multiprotein complex and to even gain insight into relative stoichiometries of proteins within the complex. In this unit, strategies, tools, and background for designing and interpreting acceptor-photobleaching FRET experiments in cells are described.

Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP-fusion proteins and multimode FRET microscopy.

Summary Multimode fluorescence resonance energy transfer (FRET) microscopy was applied to study the plasma membrane organization using different lipidated green fluorescent protein (GFP)-fusion proteins co-expressed in cowpea protoplasts. Cyan fluorescent protein (CFP) was fused to the hyper variable region of a small maize GTPase (ROP7) and yellow fluorescent protein (YFP) was fused to the N-myristoylation motif of the calcium-dependent protein kinase 1 (LeCPK1) of tomato. Upon co-expressing in cowpea protoplasts a perfect co-localization at the plasma membrane of the constructs was observed. Acceptor-photobleaching FRET microscopy indicated a FRET efficiency of 58% in protoplasts co-expressing CFP-Zm7hvr and myrLeCPK1-YFP, whereas no FRET was apparent in protoplasts co-expressing CFP-Zm7hvr and YFP. Fluorescence spectral imaging microscopy (FSPIM) revealed, upon excitation at 435 nm, strong YFP emission in the fluorescence spectra of the protoplasts expressing CFP-Zm7hvr and myrLeCPK1-YFP. Also, fluorescence lifetime imaging microscopy (FLIM) analysis indicated FRET because the CFP fluorescence lifetime of CFP-Zm7hvr was reduced in the presence of myrLeCPK1-YFP. A FRET fluorescence recovery after photobleaching (FRAP) analysis on a partially acceptor-bleached protoplast co-expressing CFP-Zm7hvr and myrLeCPK1-YFP revealed slow requenching of the CFP fluorescence in the acceptor-bleached area upon diffusion of unbleached acceptors into this area. The slow exchange of myrLeCPK1-YFP in the complex with CFP-Zm7hvr reflects a relatively high stability of the complex. Together, the FRET data suggest the existence of plasma membrane lipid microdomains in cowpea protoplasts.