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PubMed2005

A new pair for inter- and intra-molecular FRET measurement.

Fluorescence resonance energy transfer between mutant green fluorescent proteins provides powerful means to monitor in vivo protein-protein proximity and intracellular signaling. However, the current widely applied FRET pair of this class (CFP/YFP) requires excitation by expensive UV lasers, thereby hindering FRET imaging on many confocal microscopes. Further challenges arise from the large spectral overlap of CFP/YFP emission. Another FRET pair GFP/DsRed could obviate such limitations. However, the use of DsRed as a FRET acceptor is hampered by several critical problems, including a slow and incomplete maturation and obligate tetramerization. A tandem dimer mutant of DsRed (TDimer2) has similar spectral properties as those of DsRed. The rapid maturation and non-oligomerization make TDimer2 a promising substitute for DsRed in FRET experiments. Here, we have explored the possibility of using TDimer2 as a FRET acceptor for the donor EGFP. FRET was demonstrated between the EGFP-TDimer2 chimeric fusion protein. By substituting CFP/YFP in the Ca2+-sensor cameleon with EGFP/TDimer2, dynamic changes in cytosolic free Ca2+ concentrations were observed with 488nm excitation under conventional wide-field microscopy. The EGFP/TDimer2 pair was further successfully employed to monitor inter-molecular interaction between Syntaxin and SNAP25. These results reveal EGFP/TDimer2 as a promising FRET pair in monitoring intra-molecular conformation change as well as inter-molecular interaction.

Animals↗
PubMed2003

DsRed as a potential FRET partner with CFP and GFP.

Fluorescence resonance energy transfer (FRET) between mutant green fluorescent proteins (GFP) provides powerful means to monitor in vivo protein-protein proximity and intracellular messengers. However, the leading FRET pair of this class (CFP/YFP) entails suboptimal donor excitation by Argon lasers, thereby hindering FRET imaging on many confocal microscopes. Further challenges arise from the large spectral overlap of CFP/YFP emission. By contrast, DsRed, along with other members of a growing family of red-shifted sea coral fluorophores, features spectra that could obviate such limitations, using DsRed as FRET acceptor, and GFP or CFP as donor. Nonetheless, DsRed suffers from slow chromophore maturation, which confounds quantitative FRET. Here, we develop strategies minimizing the resulting complexity: 1), Pulsed activation of inducible promoters, driving expression of DsRed-tagged molecules, yields a uniform bolus of mature fluorophore; 2), The 3(3)-FRET detection algorithm, adapted for CFP/DsRed and GFP/DsRed, proves insensitive to distortion by slow maturation. We thus show that DsRed supports strong FRET in CFP-DsRed or GFP-DsRed concatemers. These results reveal the promise of sea coral fluorophores like DsRed as FRET partners with GFP or CFP.

Algorithms↗
PubMedDate not supplied

FRET-based analysis of TRPC subunit stoichiometry.

By analogy to other cation channel subunits with six transmembrane-spanning domains, the seven members of the "classical" or "canonical" transient receptor potential channels (TRPC) family are believed to assemble into homo- or heterotetrameric complexes. These complexes have been verified by classical methods such as coimmunoprecipitation, crosslinking analysis or functional assays applying dominant negative pore mutants. More recently, fluorescence resonance energy transfer (FRET)-a measure for the close proximity of fluorescent molecules-has become instrumental in monitoring protein assembly in living cells. Here we demonstrate further possibilities and verification procedures of the FRET technology to test the assembly of ion channel subunits. Temporally and spatially resolved FRET imaging demonstrates an early assembly of TRPC subunits in the endoplasmic reticulum and the Golgi apparatus. Confocal FRET imaging verifies FRET signals over the plasma membrane at high spatial resolution. Taking advantage of the quantitative analysis of digital video imaging, we demonstrate that FRET between TRPC subunits is only poorly concentration-dependent. Moreover, a correlation between the efficiency of energy transfer and the molar ratio of the FRET donor to the acceptor was exploited to verify the tetrameric stoichiometry of TRPC complexes. Finally, we introduce a competition-FRET assay to test the ability of wild-type TRPC subunits to recruit fluorescent TRPC subunits into separate channel complexes.

Calcium Channels↗
PubMed2000

Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization.

A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method.

DNA Probes↗
PubMed2005

Fluorescence resonance energy transfer (FRET) measurement by gradual acceptor photobleaching.

Fluorescence resonance energy transfer (FRET) is an extremely effective tool to detect molecular interaction at suboptical resolutions. One of the techniques for measuring FRET is acceptor photobleaching: the increase in donor fluorescence after complete acceptor photobleaching is a measure of the FRET efficiency. However, in wide-field microscopy, complete acceptor photobleaching is difficult due to the low excitation intensities. In addition, the method is sensitive to inadvertent donor bleaching, autofluorescence and bleed-through of excitation light. In the method introduced in this paper, donor and acceptor intensities are monitored continuously during acceptor photobleaching. Subsequently, curve fitting is used to determine the FRET efficiency. The method was demonstrated on cameleon (YC2.1), a FRET-based Ca(2+) indicator, and on a CFP-YFP fusion protein expressed in HeLa cells. FRET efficiency of cameleon in the presence of 1 mm Ca(2+) was 31 +/- 3%. In the absence of Ca(2+) a FRET efficiency of 15 +/- 2% was found. A FRET efficiency of 28% was found for the CFP-YFP fusion protein in HeLa cells. Advantages of the method are that it does not require complete acceptor photobleaching, it includes correction for spectral cross-talk, donor photobleaching and autofluorescence, and is relatively simple to use on a normal wide-field microscope.

Fluorescence Resonance Energy Transfer↗
PubMed2006

Cerulean, Venus, and VenusY67C FRET reference standards.

Förster's resonance energy transfer (FRET) can be used to study protein-protein interactions in living cells. Numerous methods to measure FRET have been devised and implemented; however, the accuracy of these methods is unknown, which makes interpretation of FRET efficiency values difficult if not impossible. This problem exists due to the lack of standards with known FRET efficiencies that can be used to validate FRET measurements. The advent of spectral variants of green fluorescent protein and easy access to cell transfection technology suggests a simple solution to this problem: the development of genetic constructs with known FRET efficiencies that can be replicated with high fidelity and freely distributed. In this study, fluorescent protein constructs with progressively larger separation distances between donors and acceptors were generated and FRET efficiencies were measured using fluorescence lifetime spectroscopy, sensitized acceptor emission, and spectral imaging. Since the results from each method were in good agreement, the FRET efficiency value of each construct could be determined with high accuracy and precision, thereby justifying their use as standards.

Cell Line↗
PubMed1995

Mixed-metal fretting corrosion of Ti6Al4V and wrought cobalt alloy.

Corrosion has been reported at the modular interfaces of total joint replacement implants, but with conflicting theories as to the cause of such damage. The modular design itself leaves the interface susceptible to galvanic, crevice, or fretting corrosion, or a combination of the three. The purpose of this study was to quantify the effect of material combination on fretting corrosion of orthopedic alloys. Each test specimen consisted of a two-hole plate with spherical countersinks and two cortical bone screws. The plates and screws were made of either Ti6Al4V or wrought cobalt-chromium-molybdenum (CCM), and were tested in all mixed-metal and similar-alloy combinations. Fretting corrosion experiments were conducted for 14 days in 10% calf serum, according to ASTM F897. Corrosion damage was evaluated by weight-loss measurements, atomic absorption spectrophotometry and scanning electron microscopy analyses. The results indicated that Ti6Al4V suffered relatively severe damage when fretted against itself, as a result of adhesive galling. The extent of titanium damage was reduced considerably, however, when Ti6Al4V was fretted against wrought CCM. In contrast, there was essentially no difference in wrought CCM damage when the alloys was fretted against itself compared to fretting against Ti6Al4V. Finally, in similar-alloy combinations, Ti6Al4V suffered more severe damage than wrought CCM.

Alloys↗
PubMed2006

Effect of impact assembly on the fretting corrosion of modular hip tapers.

The goal of this study was to determine the effect of assembly load and local assembly environmental conditions on the fretting corrosion of modular femoral stem tapers. Femoral head/taper assemblies in both similar (CoCrMo/CoCrMo) and mixed (CoCrMo/Ti-6Al-4V) alloy combinations were evaluated using an electrochemical test method. Specimens were assembled under impact loading and by hand, in both wet and dry conditions. Incremental cyclic loads ranging from 89 to 5,340 N were applied at a frequency of 3 Hz in Ringer's solution at ambient temperature. During the test, both the open circuit potential (OCP) and fretting current (i(fret)) were measured using a saturated calomel electrode (SCE) and counter electrode, respectively. The results were comparable for both mixed and similar alloy couples. Decreases in OCP and increases in i(fret) (indicators of oxide film fracture and repassivation) were seen with increasing load magnitude, often occurring at loads well below those expected clinically. OCP at the 5,340 N cyclic load ranged from -30.4 to -103.7 mV versus SCE for similar alloy couples, and -19.1 to -181.4 mV versus SCE for mixed alloy couples. Mean peak fretting currents ranged from 0.84 to 1.42 microA and 1.06 to 3.12 microA for similar and mixed alloy couples, respectively. The larger current magnitudes and more negative shifts in OCP for mixed alloy couples indicate the difference in oxide film fracture behavior between titanium and cobalt alloys. The load at which OCP began to drop (onset of fretting) was dependent upon the assembly conditions for both material couples. Specimens assembled with impact loads in air showed the highest resistance to fretting. The results of this study indicate that the assembly load and the environment both play a role in the initial stability of modular hip taper connections.

Chromium Alloys↗
PubMed2004

Dual-channel photobleaching FRET microscopy for improved resolution of protein association states in living cells.

Fluorescence resonance energy transfer (FRET) from a donor-labelled molecule to an acceptor-labelled molecule is a useful, proximity-based fluorescence tool to discriminate molecular states on the surface and in the interior of cells. Most microscope-based determinations of FRET yield only a single value, the interpretation of which is necessarily model-dependent. In this paper we demonstrate two new measurements of FRET heterogeneity using selective donor photobleaching in combination with synchronous donor/acceptor detection based on either (1) full kinetic analysis of donor-detected and acceptor-detected donor photobleaching or (2) a simple time-based ratiometric approach. We apply the new methods to study the cell surface distribution of concanavalin A yielding estimates of FRET and non-FRET population distributions, as well as FRET efficiencies within the FRET populations.

Animals↗
PubMed2004

Red-edge anisotropy microscopy enables dynamic imaging of homo-FRET between green fluorescent proteins in cells.

Steady-state fluorescence anisotropy measurements can be used to detect fluorescence resonance energy transfer (FRET) between identical fluorophores (homo-FRET). However, the contribution of homo-FRET to the steady-state anisotropy must be discerned from those due to the orientational distribution and rotational diffusion, which so far has required photobleaching controls, largely precluding dynamic measurements in live cells. We describe a variation of steady-state anisotropy microscopy in which the contribution of homo-FRET is dynamically isolated from the total anisotropy by exploiting the loss of energy transfer that occurs at red-edge excitation. Excitation of enhanced green fluorescent protein (EGFP) at the red-edge of its absorption band shows the shift in the emission spectrum compared to main-band excitation that is characteristic for photo-selection of static low energy S(0)-S(1) transitions that fail to exhibit FRET. An experimental setup for steady-state fluorescent anisotropy microscopy is described that can be used to acquire anisotropy images in live cells at main-band and red-edge excitation of EGFP. We demonstrate in live cells homo-FRET suppression of protein fusion constructs that consist of two and three EGFP molecules connected by short linkers. This methodology represents a novel approach for the dynamic measurement of homo-FRET in live cells that will be of utility in the biological sciences to detect oligomerization and concentration dependent interactions between identically labeled molecules.

Anisotropy↗
PubMed2002

Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data.

We studied the fluorescence resonance energy transfer (FRET) efficiency of different donor-acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G); and a carbocyanine dye (Cy3) were covalently attached to the 5'-end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and a rhodamine derivative (JA133) were attached at modified thymidine bases in the complementary DNA strand with donor-acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donor/acceptor (D/A) distances and orientations. FRET efficiencies have been calculated from donor and acceptor fluorescence intensity as well as from time-resolved fluorescence measurements of the donor fluorescence decay. Dependent on the D/A pair and distance, additional strong fluorescence quenching of the donor is observed, which simulates lower FRET efficiencies at short distances and higher efficiencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efficiency, even at short D/A distances. In addition, the measured acceptor fluorescence intensities and lifetimes also partly show fluorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the D/A distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short D/A distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger D/A distances, we suggest that the pi-stack of the DNA double helix mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve fluorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost.

Carbocyanines↗
PubMed2006

Self-assembled pi-nanotapes as donor scaffolds for selective and thermally gated fluorescence resonance energy transfer (FRET).

Self-assembled nanotapes of a few tailor-made oligo(p-phenylenevinylene)s (OPVs) have been prepared and used as supramolecular donor scaffold to study the fluorescence resonance energy transfer (FRET) to a suitable acceptor. In nonpolar solvents, FRET occurs with nearly 63-81% efficiency, exclusively from the self-assembled OPVs to entrapped Rhodamine B, resulting in the quenching of the donor emission with concomitant formation of the acceptor emission at 625 nm. The efficiency of FRET is considerably influenced by the ability of the OPVs to form the self-assembled aggregates and hence could be controlled by structural variation of the molecules, and polarity of the solvent. Most importantly, FRET could be controlled by temperature as a result of the thermally reversible self-assembly process. The FRET efficiency was significantly enhanced (ca. 90%) in a xerogel film of the OPV1 which is dispersed with relatively less amount of the acceptor (33 mol %), when compared to that of the aggregates in dodecane gel. FRET is not efficient in polar solvents due to weak self-organization of the chromophores. These results indicate that energy transfer occurs exclusively from the self-assembled donor and not directly from the individual donor molecules. The present study illustrates that the self-assembly of chromophores facilitates temperature and solvent controlled FRET within pi-conjugated nanostructures.

Journal Article↗
PubMed2004

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.

Base Sequence↗
PubMed2004

Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing.

Fluorescence resonance energy transfer (FRET) by acceptor photobleaching is a simple but effective tool for measurements of protein-protein interactions. Until recently, it has been restricted to qualitative or relative assessments owing to the spectral bleed-through contamination resulting from fluorescence overlap between the donor and the acceptor. In this paper, we report a quantitative algorithm that combines the spectral unmixing technique with FRET by acceptor photobleaching. By spectrally unmixing the emissions before and after photobleaching, it is possible to resolve the spectral bleed-through and retrieve the FRET efficiency/interaction distance quantitatively. Using a human keratinocyte cell line transfected with cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-tagged Cx26 connexins as an example, FRET information at homotypic gap junctions is measured and compared with well-established methods. Results indicate that the new approach is sensitive, flexible, instrument independent and solely FRET dependent. It can achieve FRET estimations similar to that from a sensitized emission FRET method. This approach has a great advantage in providing the relative concentrations of the donor and the acceptor; this is, for example, very important in the comparative study of cell populations with variable expression levels.

Algorithms↗
PubMed2003

Protein localization in living cells and tissues using FRET and FLIM.

Interacting proteins assemble into molecular machines that control cellular homeostasis in living cells. While the in vitro screening methods have the advantage of providing direct access to the genetic information encoding unknown protein partners, they do not allow direct access to interactions of these protein partners in their natural environment inside the living cell. Using wide-field, confocal, or two-photon (2p) fluorescence resonance energy transfer (FRET) microscopy, this information can be obtained from living cells and tissues with nanometer resolution. One of the important conditions for FRET to occur is the overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor. As a result of spectral overlap, the FRET signal is always contaminated by donor emission into the acceptor channel and by the excitation of acceptor molecules by the donor excitation wavelength. Mathematical algorithms are required to correct the spectral bleed-through signal in wide-field, confocal, and two-photon FRET microscopy. In contrast, spectral bleed-through is not an issue in FRET/FLIM imaging because only the donor fluorophore lifetime is measured; also, fluorescence lifetime imaging microscopy (FLIM) measurements are independent of excitation intensity or fluorophore concentration. The combination of FRET and FLIM provides high spatial (nanometer) and temporal (nanosecond) resolution when compared to intensity-based FRET imaging. In this paper, we describe various FRET microscopy techniques and its application to protein-protein interactions.

Animals↗
PubMed2006

Fanciful FRET.

The validity of experiments based on Förster resonance energy transfer (FRET), an imaging technique widely used to measure protein-protein interactions in living cells, critically depends on the accurate and precise measurement of FRET efficiency. The use of FRET standards to determine FRET efficiency, and a consideration of such factors as how the abundance of FRET acceptors and the stoichiometry of donors and acceptors in a molecular complex can affect measured FRET efficiency, will enhance the usefulness with which FRET experiments can be interpreted.

Algorithms↗
PubMed2001

The fretting corrosion resistance of PVD surface-modified orthopedic implant alloys.

The objective of this study was to evaluate the fretting corrosion resistance of both modified and unmodified Ti6Al4V flats fretted against CoCr-alloy spheres in a buffered Hank's solution at 37 degrees C using an original fretting apparatus. A physical vapor deposition (PVD) cathodic arc evaporation technique was used to deposit 3-4 microm thick titanium nitride (TiN), zirconium nitride (ZrN), or amorphous carbon (AC) coatings onto the Ti6Al4V substrates. The fretting behavior of the nitride films (TiN and ZrN) was characterized by the absence of surface damage and the deposition of a Cr-rich oxide transferred from the CoCr-alloy spheres to the modified surfaces. This oxide led to a slight increase in surface roughness. Three of the six multilayered AC coatings tested exhibited extensive fretting damage and generated large, deep, wear scars. Cohesive failure of the AC coating was observed in the low contact stress areas of the fretting scars. The remaining AC-coated specimens experienced only slight polishing wear. The reason for the different behavior within the AC-coated specimens is not clear at the present time. The unmodified Ti6Al4V surfaces experienced severe surface damage consistent with the adhesive galling mechanism to which these alloys are susceptible.

Arthroplasty, Replacement, Hip↗
PubMed2006

Frequency effect in fretting wear of Co-28Cr-6Mo versus Ti-6Al-4V implant alloys.

A piezo-electrically driven fretting testing device has been constructed and fretting release and release rates have been determined with highest accuracy, using a radiotracer technique. First results on the fretting release and release rate of titanium alloy fretting pads against cobalt-chrome alloy fatigue specimens are reported. The frequency dependency of fretting release has been determined between 1 and 8 Hz and shows higher release rates for low frequencies, thus indicating that accelerated testing of materials and components of artificial joints must be analyzed extremely carefully. The present experiments under simple conditions present a base-line study for step-wise applying more complex and realistic testing conditions and for using radiotracer methods to quantify fretting release in simulated testing of artificial hip- and knee-prostheses.

Alloys↗