ATP changes the fluorescence lifetime of cyan fluorescent protein via an interaction with His148

Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescence Resonance Energy Transfer (FRET) sensors based on CFP-YFP. To get insight into this phenomenon, we employed fluorescence lifetime spectroscopy to analyze the influence of ATP on these fluorescent proteins in more detail. Using different donor and acceptor pairs we found that ATP only affected the CFP-YFP based versions. Subsequent analysis of purified monomers of the used proteins showed that ATP has a direct effect on the fluorescence lifetime properties of CFP. Since the fluorescence lifetime analysis of CFP is rather complicated by the existence of different lifetimes, we tested a variant of CFP, i.e. Cerulean, as a monomer and in our FRET constructs. Surprisingly, this CFP variant shows no ATP concentration dependent changes in the fluorescence lifetime. The most important difference between CFP and Cerulean is a histidine residue at position 148. Indeed, changing this histidine in CFP into an aspartic acid results in identical fluorescence properties as observed for the Cerulean fluorescent based FRET sensor. We therefore conclude that the changes in fluorescence lifetime of CFP are affected specifically by possible electrostatic interactions of the negative charge of ATP with the positively charged histidine at position 148. Clearly, further physicochemical characterization is needed to explain the sensitivity of CFP fluorescence properties to changes in environmental (i.e. ATP concentrations) conditions.     

Some secrets of fluorescent proteins: distinct bleaching in various mounting fluids and photoactivation of cyan fluorescent proteins at YFP-excitation

Background

The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins.

Methodology/Principal Findings

When we applied a commonly used FRET microscopy technique - the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10 - 15% after illumination at the YFP-excitation wavelength – a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor.

Conclusions/Significance

Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions.     

Quantification of protein interaction in living cells by two-photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed-through

Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful method to visualize and quantify protein-protein interaction in living cells. Unfortunately, the emission bleed-through of FPs limits the usage of this complex technique. To circumvent undesirable excitation of the acceptor fluorophore, using two-photon excitation, we searched for FRET pairs that show selective excitation of the donor but not of the acceptor fluorescent molecule. We found this property in the fluorescent cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) and YFP/mCherry FRET pairs and performed two-photon excited FRET spectral imaging to quantify protein interactions on the later pair that shows better spectral discrimination. Applying non-negative matrix factorization to unmix two-photon excited spectral imaging data, we were able to eliminate the donor bleed-through as well as the autofluorescence. As a result, we achieved FRET quantification by means of a single spectral acquisition, making the FRET approach not only easy and straightforward but also less prone to calculation artifacts. As an application of our approach, the intermolecular interaction of amyloid precursor protein and the adaptor protein Fe65 associated with Alzheimer's disease was quantified. We believe that the FRET approach using two-photon and fluorescent YFP/mCherry pair is a promising method to monitor protein interaction in living cells.     

A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells

Fluorescence resonance energy transfer (FRET) from cyan to yellow fluorescent proteins (CFP/YFP) is a well-established method to monitor protein-protein interactions or conformational changes of individual proteins. But protein functions can be perturbed by fusion of large tags such as CFP and YFP. Here we use G protein-coupled receptor (GPCR) activation in living cells as a model system to compare YFP with the small, membrane-permeant fluorescein derivative with two arsen-(III) substituents (fluorescein arsenical hairpin binder; FlAsH) targeted to a short tetracysteine sequence. Insertion of CFP and YFP into human adenosine A(2A) receptors allowed us to use FRET to monitor receptor activation but eliminated coupling to adenylyl cyclase. The CFP/FlAsH-tetracysteine system gave fivefold greater agonist-induced FRET signals, similar kinetics (time constant of 66-88 ms) and perfectly normal downstream signaling. Similar results were obtained for the mouse alpha(2A)-adrenergic receptor. Thus, FRET from CFP to FlAsH reports GPCR activation in living cells without disturbing receptor function and shows that the small size of the tetracysteine-biarsenical tag can be decisively advantageous.     

Three-Dimensional Reconstruction of Three-Way FRET Microscopy Improves Imaging of Multiple Protein-Protein Interactions

Fluorescence resonance energy transfer (FRET) microscopy is a powerful tool for imaging the interactions between fluorescently tagged proteins in two-dimensions. For FRET microscopy to reach its full potential, it must be able to image more than one pair of interacting molecules and image degradation from out-of-focus light must be reduced. Here we extend our previous work on the application of maximum likelihood methods to the 3-dimensional reconstruction of 3-way FRET interactions within cells. We validated the new method (3D-3Way FRET) by simulation and fluorescent protein test constructs expressed in cells. In addition, we improved the computational methods to create a 2-log reduction in computation time over our previous method (3DFSR). We applied 3D-3Way FRET to image the 3D subcellular distributions of HIV Gag assembly. Gag fused to three different FPs (CFP, YFP, and RFP), assembled into viral-like particles and created punctate FRET signals that become visible on the cell surface when 3D-3Way FRET was applied to the data. Control experiments in which YFP-Gag, RFP-Gag and free CFP were expressed, demonstrated localized FRET between YFP and RFP at sites of viral assembly that were not associated with CFP. 3D-3Way FRET provides the first approach for quantifying multiple FRET interactions while improving the 3D resolution of FRET microscopy data without introducing bias into the reconstructed estimates. This method should allow improvement of widefield, confocal and superresolution FRET microscopy data.     

Enhanced dynamic range in a genetically encoded Ca2+ sensor

Genetically encoded fluorescence resonance energy transfer (FRET) indicators are powerful tools for real-time detection of second messenger molecules and activation of signal proteins. However, these fluorescent protein-based sensors typically display marginal FRET efficiency. To improve their FRET efficiency for optical imaging and screening, we developed a number of fluorescent protein mutants based on cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). To improve FRET ratios, which were initially within a narrow dynamic range, we used DNA shuffling to develop a new FRET pair called 3xCFP/Venus. The optimized 3xCFP/Venus pair exhibited higher FRET ratios than CyPet/YPet, which has one of the greatest dynamic ranges of protein-based FRET pairs. We converted this FRET pair to a Ca(2+) FRET indicators using circular permutation Venus (cpVenus) linked with 3xCFP to form 3xCFP/cpVenus, which displayed an ～11-fold change in dynamic range in response to Ca(2+) binding. The enhanced dynamic range for Ca(2+) concentration detection using 3xCFP/cpVenus was confirmed in PC12 cells using previously established indicators (TN-XXL, ECFP/cpCitrine). To our knowledge, this FRET pair displays the largest dynamic range so far among genetically-encoded sensors, and can be used for sensitive FRET detection.     

Enhanced dynamic range in a genetically encoded Ca sensor

Genetically encoded fluorescence resonance energy transfer (FRET) indicators are powerful tools for real-time detection of second messenger molecules and activation of signal proteins. However, these fluorescent protein-based sensors typically display marginal FRET efficiency. To improve their FRET efficiency for optical imaging and screening, we developed a number of fluorescent protein mutants based on cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). To improve FRET ratios, which were initially within a narrow dynamic range, we used DNA shuffling to develop a new FRET pair called 3xCFP/Venus. The optimized 3xCFP/Venus pair exhibited higher FRET ratios than CyPet/YPet, which has one of the greatest dynamic ranges of protein-based FRET pairs. We converted this FRET pair to a Ca²⁺ FRET indicators using circular permutation Venus (cpVenus) linked with 3xCFP to form 3xCFP/cpVenus, which displayed an ∼11-fold change in dynamic range in response to Ca²⁺ binding. The enhanced dynamic range for Ca²⁺ concentration detection using 3xCFP/cpVenus was confirmed in PC12 cells using previously established indicators (TN-XXL, ECFP/cpCitrine). To our knowledge, this FRET pair displays the largest dynamic range so far among genetically-encoded sensors, and can be used for sensitive FRET detection.     

Evaluation of the CFP-substrate-YFP system for protease studies: advantages and limitations

A protease can be defined as an enzyme capable of hydrolyzing peptide bonds. Thus, characterization of a protease involves identification of target peptide sequences, measurement of activities toward these sequences, and determination of kinetic parameters. Biological protease substrates based on fluorescent protein pairs, which allow for use of fluorescence resonance energy transfer (FRET), have been recently developed for in vivo protease activity detection and represent a very interesting alternative to chemical substrates for in vitro protease characterization. Here, we analyze a FRET system consisting of cyan and yellow fluorescent proteins (CFP and YFP, respectively), which are fused by a peptide linker serving as protease substrate. Conditions for CFP-YFP fusion protein production in Escherichia coli and purification of proteins were optimized. FRET between CFP and YFP was found to be optimum at a pH between 5.5 and 10.0, at low concentrations of salt and a temperature superior to 25 degrees C. For efficient FRET to occur, the peptide linker between CFP and YFP can measure up to 25 amino acids. The CFP-substrate-YFP system demonstrated a high degree of resistance to nonspecific proteolysis, making it suitable for enzyme kinetic analysis. As with chemical substrates, substrate specificity of CFP-substrate-YFP proteins was tested towards different proteases and kcat/Km values were calculated.     

A genetically encoded Förster resonance energy transfer biosensor for two-photon excitation microscopy

Pippi (phosphatidyl inositol phosphate indicator) is a biosensor based on the principle of FRET (F?rster resonance energy transfer), which consists of a pair of fluorescent proteins, CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein), the PH domain sandwiched between them, and K-Ras C-terminal sequence for plasma membrane localization. Due to marked cross-excitation of YFP with the conditions used to excite CFP, initial FRET images obtained by TPE (two-photon excitation) microscopy suffered from low signal-to-noise ratio, hampering the observation of lipids in three-dimensional structures. To solve this problem, YFP and CFP in the original Pippi-PI(3,4)P(2) was replaced by sREACh (super resonance energy accepting chromoprotein) and mTFP1 (monomeric teal fluorescent protein), respectively. The biosensor was also fused with an internal control protein, mKeima, where Keima/mTFP1 indicates the FRET efficiency, and indeed epidermal growth factor stimulation increased Keima/mTFP1 in HeLa cells. This biosensor successfully showed PI(3,4)P(2) accumulation to the lateral membrane in the MDCK cyst cultured in a three-dimensional environment. Furthermore, other FRET-based biosensors for PIP(3) distribution and for tyrosine kinase activity were developed based on this method, suggesting its broad application for visualizing signal transduction events with TPE microscopy.     

Fluorescence resonance energy transfer analysis of cell surface receptor interactions and signaling using spectral variants of the green fluorescent protein

BACKGROUND: Fluorescence resonance energy transfer (FRET) is a powerful technique for measuring molecular interactions at Angstrom distances. We present a new method for FRET that utilizes the unique spectral properties of variants of the green fluorescent protein (GFP) for large-scale analysis by flow cytometry. METHODS: The proteins of interest are fused in frame separately to the cyan fluorescent protein (CFP) or the yellow fluorescent protein (YFP). FRET between these differentially tagged fusion proteins is analyzed using a dual-laser FACSVantage cytometer. RESULTS: We show that homotypic interactions between individual receptor chains of tumor necrosis factor receptor (TNFR) family members can be detected as FRET from CFP-tagged receptor chains to YFP-tagged receptor chains. Noncovalent molecular complexation can be detected as FRET between fusions of CFP and YFP to either the intracellular or extracellular regions of the receptor chains. The specificity of the assay is demonstrated by the absence of FRET between heterologous receptor pairs that do not biochemically associate with each other. Interaction between a TNFR-like receptor (Fas/CD95/Apo-1) and a downstream cytoplasmic signaling component (FADD) can also be demonstrated by flow cytometric FRET analysis. CONCLUSIONS: The utility of spectral variants of GFP in flow cytometric FRET analysis of membrane receptors is demonstrated. This method of analyzing FRET allows probing of noncovalent molecular interactions that involve both the intracellular and extracellular regions of membrane proteins as well as proteins within the cells. Unlike biochemical methods, FRET allows the quantitative determination of noncovalent molecular associations at Angstrom level in living cells. Moreover, flow cytometry allows quantitative analyses to be carried out on a cell-by-cell basis on large number of cells. Published 2001 Wiley-Liss, Inc.     

A genetically encoded Förster resonance energy transfer biosensor for two-photon excitation microscopy

Pippi (phosphatidyl inositol phosphate indicator) is a biosensor based on the principle of FRET (Förster resonance energy transfer), which consists of a pair of fluorescent proteins, CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein), the PH domain sandwiched between them, and K-Ras C-terminal sequence for plasma membrane localization. Due to marked cross-excitation of YFP with the conditions used to excite CFP, initial FRET images obtained by TPE (two-photon excitation) microscopy suffered from low signal-to-noise ratio, hampering the observation of lipids in three-dimensional structures. To solve this problem, YFP and CFP in the original Pippi-PI(3,4)P₂ was replaced by sREACh (super resonance energy accepting chromoprotein) and mTFP1 (monomeric teal fluorescent protein), respectively. The biosensor was also fused with an internal control protein, mKeima, where Keima/mTFP1 indicates the FRET efficiency, and indeed epidermal growth factor stimulation increased Keima/mTFP1 in HeLa cells. This biosensor successfully showed PI(3,4)P₂ accumulation to the lateral membrane in the MDCK cyst cultured in a three-dimensional environment. Furthermore, other FRET-based biosensors for PIP₃ distribution and for tyrosine kinase activity were developed based on this method, suggesting its broad application for visualizing signal transduction events with TPE microscopy.     

APPL Proteins FRET at the BAR: Direct Observation of APPL1 and APPL2 BAR Domain-Mediated Interactions on Cell Membranes Using FRET Microscopy

Background

Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR) domain, a central pleckstrin homology (PH) domain, and a C-terminal phosphotyrosine binding (PTB) domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported.

Methodology

Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET) experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP) FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species.

Conclusions

All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2) and heterotypic (i.e., APPL1-APPL2) manner on curved cell membranes. Furthermore, the results of our experiments did not show photoconversion of YFP into a CFP-like species following photobleaching, supporting the use of CFP donor/YFP acceptor FRET pairs in acceptor photobleaching studies.     

High-precision FLIM-FRET in fixed and living cells reveals heterogeneity in a simple CFP-YFP fusion protein

We have used widefield photon-counting FLIM to study FRET in fixed and living cells using control FRET pairs. We have studied fixed mammalian cells expressing either cyan fluorescent protein (CFP) or a fusion of CFP and yellow fluorescent protein (YFP), and living fungal cells expressing either Cerulean or a Cerulean-Venus fusion protein. We have found the fluorescence behaviour to be essentially identical in the mammalian and fungal cells. Importantly, the high-precision FLIM data is able to reproducibly resolve multiple fluorescence decays, thereby revealing new information about the fraction of the protein population that undergoes FRET and reducing error in the measurement of donor-acceptor distances. Our results for this simple control system indicate that the in vivo FLIM-FRET studies of more complex protein-protein interactions would benefit greatly from such quantitative measurements.     

Detecting protein-protein interaction in live yeast by flow cytometry.

BACKGROUND: The yeast Saccharomyces cerevisiae is the most commonly used organism for studying protein- protein interactions. In this report we demonstrate the use of flow cytometry in observing fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent fusion proteins (CFP and YFP, respectively) as a marker for protein interaction in live yeast cells. Probability binning is also employed to provide a statistical confirmation of our observations. METHODS: We coexpressed CFP and YFP fusions containing the N-terminal transmembrane domain (NTM) of Tom70p in yeast and analyzed FRET in live cells with a multilaser flow cytometer. The Tom70p NTM was previously shown to be sufficient for mitochondrial localization and protein-protein interaction (Millar and Shore, 1994, J Biol Chem 269:12229-12232). RESULTS: FRET was observed only in cells that expressed CFP and YFP fusions that each contained the wild-type NTM. The introduction of mutations previously shown to disrupt NTM interaction eliminated FRET. Probability binning confirmed that differences between the FRET channels of experimental and control samples were statistically and physiologically significant. CONCLUSION: Flow cytometric analysis of FRET in yeast is a powerful technique for studying protein-protein interactions. The use of flow cytometry allows FRET data to be gathered from a large number of individual cells, thus providing important advantages unavailable to other techniques. Its application to yeast presents a new method to a popular system widely used in proteomic studies.     

Phospholemman phosphorylation alters its fluorescence resonance energy transfer with the Na/K-ATPase pump

Phospholemman (PLM) or FXYD1 is a major cardiac myocyte phosphorylation target upon adrenergic stimulation. Prior immunoprecipitation and functional studies suggest that phospholemman associates with the Na/K-pump (NKA) and mediates adrenergic Na/K-pump regulation. Here, we tested whether the NKA-PLM interaction is close enough to allow fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent (CFP/YFP) fusion proteins of Na/K pump and phospholemman and whether phospholemman phosphorylation alters such FRET. Co-expressed NKA-CFP and PLM-YFP in HEK293 cells co-localized in the plasma membrane and exhibited robust FRET. Selective acceptor photobleach increased donor fluorescence (F(CFP)) by 21.5 +/- 4.1% (n = 13), an effect nearly abolished when co-expressing excess phospholemman lacking YFP. Activation of protein kinase C or A progressively and reversibly decreased FRET assessed by either the fluorescence ratio (F(YFP)/F(CFP)) or the enhancement of donor fluorescence after acceptor bleach. After protein kinase C activation, forskolin did not further reduce FRET, but after forskolin pretreatment, protein kinase C could still reduce FRET. This agreed with phospholemman phosphorylation measurements: by protein kinase C at both Ser-63 and Ser-68, but by protein kinase A only at Ser-68. Expression of PLM-YFP and PLM-CFP resulted in even stronger FRET than for NKA-PLM (F(CFP) increased by 37 +/- 1% upon YFP photobleach), and this FRET was enhanced by phospholemman phosphorylation, consistent with phospholemman multimerization. Co-expressed PLM-CFP and Na/Ca exchange-YFP were highly membrane co-localized, but FRET was undetectable. We conclude that phospholemman and Na/K-pump are in very close proximity (FRET occurs) and that phospholemman phosphorylation alters the interaction of Na/K-pump and phospholemman.     

Stable SNARE complex prior to evoked synaptic vesicle fusion revealed by fluorescence resonance energy transfer

Although it is clear that soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complex plays an essential role in synaptic vesicle fusion, the dynamics of SNARE assembly during vesicle fusion remain to be determined. In this report, we employ fluorescence resonance energy transfer technique to study the formation of SNARE complexes. Donor/acceptor pair variants of green fluorescent protein (GFP), cyan fluorescent protein (CFP), and yellow fluorescent protein (YFP) are fused with the N termini of SNAP-25 and synaptobrevin, respectively. In vitro assembly of SNARE core complex in the presence of syntaxin shows strong fluorescence resonance energy transfer (FRET) between the CFP-SNAP-25 and YFP-synaptobrevin. Under the same conditions, CFP fused to the C terminus of SNAP-25, and YFP- synaptobrevin have no FRET. Adenovirus-mediated gene transfer is used to express the fusion proteins in PC12 cells and cultured rat cerebellar granule cells. Strong FRET is associated with neurite membranes and vesicular structures in PC12 cells co-expressing CFP-SNAP-25 and YFP-synaptobrevin. In cultured rat cerebellar granule cells, FRET between CFP-SNAP-25 and YFP-synaptobrevin is mostly associated with sites presumed to be synaptic junctions. Neurosecretion in PC12 cells initiated by KCl depolarization leads to an increase in the extent of FRET. These results demonstrate that significant amounts of stable SNARE complex exist prior to evoked synaptic vesicle fusion and that the assembly of SNARE complex occurs during vesicle docking/priming stage. Moreover, it demonstrates that FRET can be used as an effective tool for investigating dynamic SNARE interactions during synaptic vesicle fusion.     

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.     

Regulatory activation is accompanied by movement in the C terminus of the Na-K-Cl cotransporter (NKCC1)

The Na-K-Cl cotransporter (NKCC1) is expressed in most vertebrate cells and is crucial in the regulation of cell volume and intracellular chloride concentration. To study the structure and function of NKCC1, we tagged the transporter with cyan (CFP) and yellow (YFP) fluorescent proteins at two sites within the C terminus and measured fluorescence resonance energy transfer (FRET) in stably expressing human embryonic kidney cell lines. Both singly and doubly tagged NKCC1s were appropriately produced, trafficked to the plasma membrane, and exhibited (86)Rb transport activity. When both fluorescent probes were placed within the same C terminus of an NKCC1 transporter, we recorded an 11% FRET decrease upon activation of the transporter. This result clearly demonstrates movement of the C terminus during the regulatory response to phosphorylation of the N terminus. When we introduced CFP and YFP separately in different NKCC1 constructs and cotransfected these in HEK cells, we observed FRET between dimer pairs, and the fractional FRET decrease upon transporter activation was 46%. Quantitatively, this indicates that the largest FRET-signaled movement is between dimer pairs, an observation supported by further experiments in which the doubly tagged construct was cotransfectionally diluted with untagged NKCC1. Our results demonstrate that regulation of NKCC1 is accompanied by a large movement between two positions in the C termini of a dimeric cotransporter. We suggest that the NKCC1 C terminus is involved in transport regulation and that dimerization may play a key structural role in the regulatory process. It is anticipated that when combined with structural information, our findings will provide a model for understanding the conformational changes that bring about NKCC1 regulation.     

Simultaneous Live Cell Imaging Using Dual FRET Sensors with a Single Excitation Light

Fluorescence resonance energy transfer (FRET) between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca²⁺ and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility.