Changes In Fluorescence Resonance Energy Transfer Research Articles

Transgenic Mice for cGMP Imaging

Cyclic GMP (cGMP) is an important intracellular signaling molecule in the cardiovascular system, but its spatiotemporal dynamics in vivo is largely unknown. To generate and characterize transgenic mice expressing the fluorescence resonance energy transfer-based ratiometric cGMP sensor, cGMP indicator with an EC50 of 500 nmol/L (cGi500), in cardiovascular tissues. Mouse lines with smooth muscle-specific or ubiquitous expression of cGi500 were generated by random transgenesis using an SM22α promoter fragment or by targeted integration of a Cre recombinase-activatable expression cassette driven by the cytomegalovirus early enhancer/chicken β-actin/β-globin promoter into the Rosa26 locus, respectively. Primary smooth muscle cells isolated from aorta, bladder, and colon of cGi500 mice showed strong sensor fluorescence. Basal cGMP concentrations were < 100 nmol/L, whereas stimulation with cGMP-elevating agents such as 2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium salt (DEA/NO) or the natriuretic peptides, atrial natriuretic peptide, and C-type natriuretic peptide evoked fluorescence resonance energy transfer changes corresponding to cGMP peak concentrations of ≈ 3 µmol/L. However, different types of smooth muscle cells had different sensitivities of their cGMP responses to DEA/NO, atrial natriuretic peptide, and C-type natriuretic peptide. Robust nitric oxide-induced cGMP transients with peak concentrations of ≈ 1 to > 3 µmol/L could also be monitored in blood vessels of the isolated retina and in the cremaster microcirculation of anesthetized mice. Moreover, with the use of a dorsal skinfold chamber model and multiphoton fluorescence resonance energy transfer microscopy, nitric oxide-stimulated vascular cGMP signals associated with vasodilation were detected in vivo in an acutely untouched preparation. These cGi500 transgenic mice permit the visualization of cardiovascular cGMP signals in live cells, tissues, and mice under normal and pathological conditions or during pharmacotherapy with cGMP-elevating drugs.

Single-Molecule FRET with Total Internal Reflection Microscopy

Single-molecule (sm) fluorescence detection is a powerful method for studying biological events without time and population averaging. Förster (fluorescence) resonance energy transfer (FRET) is a spectroscopic technique in which the efficiency of energy transfer from donor to acceptor molecules is used to determine distances between molecules in the 30-80 Å range. Structural changes in biological molecules or relative motion between two interacting molecules can be detected by a change in FRET. This article focuses primarily on smFRET based on total internal reflection (TIR) microscopy. It begins with discussions of dye choice and labeling of nucleic acids and proteins. These are followed by information on surface preparation and data acquisition. Various methods of data analysis are then presented, as is information on setting up TIR microscopy, both the objective and the prism types.

Prism-Type Total Internal Reflection Microscopy for Single-Molecule FRET

Single-molecule (sm) fluorescence detection is a powerful method for studying biological events without time and population averaging. Förster (fluorescence) resonance energy transfer (FRET) is a spectroscopic technique in which the efficiency of energy transfer from donor to acceptor molecules is used to determine distances between molecules in the 30-80 Å range. Structural changes in biological molecules or relative motion between two interacting molecules can be detected by a change in FRET. A variant of smFRET is based on total internal reflection (TIR) microscopy, which can be set up in two ways, either using an oil-immersion (objective-type) or a water-immersion (prism-type) lens. This protocol describes the setup for prism-type TIR microscopy (excitation and emission), which has been a major imaging tool for smFRET since it was first introduced.

Objective-Type Total Internal Reflection Microscopy (Excitation) for Single-Molecule FRET

Single-molecule (sm) fluorescence detection is a powerful method for studying biological events without time and population averaging. Förster (fluorescence) resonance energy transfer (FRET) is a spectroscopic technique in which the efficiency of energy transfer from donor to acceptor molecules is used to determine distances between molecules in the 30-80 Å range. Structural changes in biological molecules or relative motion between two interacting molecules can be detected by a change in FRET. A variant of smFRET is based on total internal reflection (TIR) microscopy, which can be set up in two ways, either using an oil-immersion (objective-type) or a water-immersion (prism-type) lens. This protocol describes objective-type TIR microscopy (excitation), including setup of the laser beam and epifluorescence microscopy, and conversion of the latter into TIR.

Objective-Type Total Internal Reflection Microscopy (Emission) for Single-Molecule FRET

Single-molecule (sm) fluorescence detection is a powerful method for studying biological events without time and population averaging. Förster (fluorescence) resonance energy transfer (FRET) is a spectroscopic technique in which the efficiency of energy transfer from donor to acceptor molecules is used to determine distances between molecules in the 30-80 Å range. Structural changes in biological molecules or relative motion between two interacting molecules can be detected by a change in FRET. A variant of smFRET is based on total internal reflection (TIR) microscopy, which can be set up in two ways, either using an oil-immersion (objective-type) or a water-immersion (prism-type) lens. This protocol describes the preparation of a fluorescent bead sample and the setup for objective-type TIR microscopy (emission).

Imaging and Identifying Impurities in Single-Molecule FRET Studies: Figure 1.

Single-molecule (sm) Förster (fluorescence) resonance energy transfer (FRET) is a spectroscopic technique in which the efficiency of energy transfer from donor to acceptor molecules is used to determine distances between molecules in the 30-80 Å range. Structural changes in biological molecules or relative motion between two interacting molecules can be detected by a change in FRET. To study the conformational changes of individual molecules over extended time periods, the molecules must be immobilized on a coated surface that allows highly specific tethering of DNA, RNA, or protein. Nevertheless, there are always some fluorescent impurities on the surface and, without experience in sm imaging, it is often difficult to distinguish molecules of interest from impurities. This protocol describes the use of an imaging buffer that enhances the photostability of cyanine dyes used for smFRET, with emphasis on distinguishing molecules of interest from impurities.

Preparing Sample Chambers for Single-Molecule FRET

Single-molecule (sm) fluorescence detection is a powerful method for studying biological events without time and population averaging. Förster (fluorescence) resonance energy transfer (FRET) is a spectroscopic technique in which the efficiency of energy transfer from donor to acceptor molecules is used to determine distances between molecules in the 30-80 Å range. Structural changes in biological molecules or relative motion between two interacting molecules can be detected by a change in FRET. A variant of smFRET is based on total internal reflection (TIR) microscopy, which can be set up in two ways, either using an oil-immersion (objective-type) or a water-immersion (prism-type) lens. To study the conformational changes of individual molecules over extended time periods, molecules must be localized in space. This protocol describes the preparation of sample chambers with either bovine serum albumin (BSA)- or polyethylene glycol (PEG)-coated slides to which single molecules can be tethered for use in FRET studies.

2-Color Calcium Pump Reveals Closure of the Cytoplasmic Headpiece with Calcium Binding

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) undergoes conformational changes while transporting calcium, but the details of the domain motions are still unclear. The objective of the present study was to measure distances between the cytoplasmic domains of SERCA2a in order to reveal the magnitude and direction of conformational changes. Using fluorescence microscopy of live cells, we measured intramolecular fluorescence resonance energy transfer (FRET) from a donor fluorescent protein fused to the SERCA N-terminus to an acceptor fluorescent protein fused to either the N-, P-, or transmembrane domain. The “2-color” SERCA constructs were catalytically active as indicated by ATPase activity in vitro and Ca uptake in live cells. All constructs exhibited dynamic FRET changes in response to the pump ligands calcium and thapsigargin (Tg). These FRET changes were quantified as an index of SERCA conformational changes. Intramolecular FRET decreased with Tg for the two N-domain fusion sites (at residue 509 or 576), while the P- (residue 661) and TM-domain (C-terminus) fusions showed increased FRET with Tg. The magnitude of the Tg-dependent conformational change was not decreased by coexpression of phospholamban (PLB), nor did PLB slow the kinetics of Tg binding. FRET in ionophore-permeabilized cells was lower in EGTA than in saturating calcium for all constructs, indicating a decrease in domain separation distance with the structural transition from E2 (Ca-free) to E1 (Ca-bound). The data suggest closure of the cytoplasmic headpiece with Ca-binding. The present results provide insight into the structural dynamics of the Ca-ATPase. In addition, the 2-color SERCA constructs developed for this study may be useful for evaluating candidate small molecule regulators of Ca uptake activity.

High-Throughput Examination of Fluorescence Resonance Energy Transfer-Detected Metal-Ion Response in Mammalian Cells

Fluorescence resonance energy transfer (FRET)-based genetically encoded metal-ion sensors are important tools for studying metal-ion dynamics in live cells. We present a time-resolved microfluidic flow cytometer capable of characterizing the FRET-based dynamic response of metal-ion sensors in mammalian cells at a throughput of 15 cells/s with a time window encompassing a few milliseconds to a few seconds after mixing of cells with exogenous ligands. We have used the instrument to examine the cellular heterogeneity of Zn(2+) and Ca(2+) sensor FRET response amplitudes and demonstrated that the cluster maps of the Zn(2+) sensor FRET changes resolve multiple subpopulations. We have also measured the in vivo sensor response kinetics induced by changes in Zn(2+) and Ca(2+) concentrations. We observed an ∼30 fold difference between the extracellular and intracellular sensors.

The Affinity of the Cardiac Na+-Ca2+ Exchanger to Regulatory Ca2+ is Decreased by Cytoplasmic Mg2+

The Na+-Ca2+ exchanger (NCX) is present in many tissues but is particularly abundant in the heart where it controls contractility. NCX exchanges three Na+ for one Ca2+ and regulates intracellular Ca2+. In addition to being transported, cytoplasmic Ca2+ activates NCX by binding to two Ca2+ domains, CBD1 and CBD2. Previously, we characterized the Ca2+-dependent structural rearrangements of the isolated Ca2+-binding domains and the full-length exchanger using Fluorescence Resonance Energy Transfer (FRET). FRET changes of NCX occurred with Kd's of activation in the low nM range (Ca2+ concentrations of 20-40 nM). This suggests that at diastolic Ca2+ levels (∼100 nM) NCX may be already fully activated. We examined the effect of Mg2+ on NCX Ca2+ regulation to determine if Mg2+ shifts the Ca activation levels towards more physiological concentrations. Monitoring FRET in real time of both the individual and conjoined CBD's and the full length NCX allowed us to determine Ca2+ affinities in the presence or absence of 1 mM Mg. The full-length exchanger, CBD1, and the conjunct CBD1-CBD2 (CBD12) exhibited a decrease in the apparent affinity for cytoplasmic Ca2+ in the presence of Mg2+. In contrast, the sensitivity of CBD2 to cytoplasmic Ca2+ increased in the presence of Mg2+. The results are consistent with in vivo data as expression of a plasma membrane targeted CBD12 in neonatal cardiac myocytes shows changes in FRET during spontaneous contractions. Our results indicate that the exchanger can indeed sense changes in subsarcolemmal Ca2+ in the physiological range (100-200 nM). Moreover, since the concentration of cytoplasmic Mg2+ is altered during heart failure, these results suggest that NCX may be differentially modulated in pathological and physiological conditions.

Dynamic force-induced direct dissociation of protein complexes in a nuclear body in living cells

Despite past progress in understanding mechanisms of cellular mechanotransduction, it is unclear whether a local surface force can directly alter nuclear functions without intermediate biochemical cascades. Here we show that a local dynamic force via integrins results in direct displacements of coilin and SMN proteins in Cajal bodies and direct dissociation of coilin-SMN associated complexes. Spontaneous movements of coilin increase more than those of SMN in the same Cajal body after dynamic force application. Fluorescence resonance energy transfer changes of coilin-SMN depend on force magnitude, an intact F-actin, cytoskeletal tension, Lamin A/C, or substrate rigidity. Other protein pairs in Cajal bodies exhibit different magnitudes of fluorescence resonance energy transfer. Dynamic cyclic force induces tiny phase lags between various protein pairs in Cajal bodies, suggesting viscoelastic interactions between them. These findings demonstrate that dynamic force-induced direct structural changes of protein complexes in Cajal bodies may represent a unique mechanism of mechanotransduction that impacts on nuclear functions involved in gene expression.

Decrease in phosphatidylinositol 4,5‐bisphosphate levels mediates desensitization of the cold sensor TRPM8 channels

The activity of the cold- and menthol-activated transient receptor potential melastatin 8 (TRPM8) channels diminishes over time in the presence of extracellular Ca(2+), a phenomenon referred to as desensitization or adaptation. Here we show that activation of TRPM8 by cold or menthol evokes a decrease in cellular phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] levels. The decrease in PtdIns(4,5)P(2) levels was accompanied by increased inositol 1,4,5 trisphosphate (InsP(3)) production, and was inhibited by loading the cells with the Ca(2+) chelator BAPTA-AM, showing that it was the consequence of the activation of phospholipase C (PLC) by increased intracellular Ca(2+) concentrations. PtdIns(4,5)P(2) hydrolysis showed excellent temporal correlation with current desensitization in simultaneous patch clamp and fluorescence-based PtdIns(4,5)P(2) level measurements. Intracellular dialysis of PtdIns(4,5)P(2) inhibited desensitization both in native neuronal and recombinant TRPM8 channels. PtdIns(4)P, the precursor of PtdIns(4,5)P(2), did not inhibit desensitization, consistent with its minimal effect in excised patches. Omission of MgATP from the intracellular solution accelerated desensitization, and MgATP reactivated TRPM8 channels in excised patches in a phosphatidylinositol 4-kinase (PI4K)-dependent manner. PLC-independent depletion of PtdIns(4,5)P(2) using a voltage-sensitive phosphatase (ci-VSP) inhibited TRPM8 currents, and omission of ATP from the intracellular solution inhibited recovery from this inhibition. Inhibitors of PKC had no effect on the kinetics of desensitization. We conclude that Ca(2+) influx through TRPM8 activates a Ca(2+)-sensitive PLC isoform, and the resulting depletion of PtdIns(4,5)P(2) plays a major role in desensitization of both cold and menthol responses.

G‐PKDrep‐live, a genetically encoded FRET reporter to measure PKD activity at the trans‐Golgi‐network

The serine/threonine protein kinase D (PKD) is recruited to the trans-Golgi-network (TGN) by interaction with diacylglycerol (DAG) and Arf1 and promotes the fission of vesicles containing cargo destined for the plasma membrane. PKD activation is mediated by PKC(-induced phosphorylation. However, signaling pathways that activate PKD specifically at the TGN are only poorly characterized. Recently we created G-PKDrep, a genetically encoded fluorescent reporter for PKD activity at the TGN in fixed cells. To establish a reporter useful for monitoring Golgi-specific PKD activity in living cells we now refined G-PKDrep to generate G-PKDrep-live. Specifically, phosphorylation of G-PKDrep-live expressed in mammalian cells results in changes of fluorescence resonance energy transfer (FRET), and allows for indirect imaging of PKD activity. In a proof-of-principle experiment using phorbolester treatment, we demonstrate the reporter's capability to track rapid activation of PKD at the TGN. Furthermore, activation-induced FRET changes are reversed by treatment with PKD-specific pharmacological inhibitors. Thus, the newly developed reporter G-PKDrep-live is a suitable tool to visualize dynamic changes in PKD activity at the TGN in living cells. See accompanying commentary by Gautam DOI: 10.1002/biot.201100424.

Phospholamban Binds with Differential Affinity to Calcium Pump Conformers

To investigate the mechanism of regulation of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) by phospholamban (PLB), we expressed Cerulean-SERCA and yellow fluorescent protein (YFP)-PLB in adult rabbit ventricular myocytes using adenovirus vectors. SERCA and PLB were localized in the sarcoplasmic reticulum and were mobile over multiple sarcomeres on a timescale of tens of seconds. We also observed robust fluorescence resonance energy transfer (FRET) from Cerulean-SERCA to YFP-PLB. Electrical pacing of cardiac myocytes elicited cytoplasmic Ca(2+) elevations, but these increases in Ca(2+) produced only modest changes in SERCA-PLB FRET. The data suggest that the regulatory complex is not disrupted by elevations of cytosolic calcium during cardiac contraction (systole). This conclusion was also supported by parallel experiments in heterologous cells, which showed that FRET was reduced but not abolished by calcium. Thapsigargin also elicited a small decrease in PLB-SERCA binding affinity. We propose that PLB is not displaced from SERCA by high calcium during systole, and relief of functional inhibition does not require dissociation of the regulatory complex. The observed modest reduction in the affinity of the PLB-SERCA complex with Ca(2+) or thapsigargin suggests that the binding interface is altered by SERCA conformational changes. The results are consistent with multiple modes of PLB binding or alternative binding sites.

Synaptotagmin 1 drives Trans SNARE Zippering with Ca2+

In neurons, Synaptotagmin 1 (Syt1) is a major Ca2++-sensor that evokes vesicle fusion, a necessary event for the neurotransmitter release, in response to the Ca2++ signal. Although Syt1 is known to bind to the core fusion machinery SNAREpin the mechanism by which Syt1 regulates vesicle fusion is not known. Here we used site-specific fluorescence resonance energy transfer (FRET) to investigate the effects of Syt1 and Ca2+ on formation of trans SNAREpin between the complimentary SNAREs reconstituted to proteoliposomes. When the FRET pairs were attached at the N-termini of SNAREs C2AB, a soluble version of Syt1, had virtually no effect on the rate of the FRET change, indicating Syt1 does not interfere with the initial stage of SNARE complex formation that happens at the membrane-distal region. However, when the FRET pairs were placed at the membrane proximal region of the SNAREs C2AB accelerated the FRET change significantly in the presence of Ca2++, indicating C2AB/Ca2+ promotes the transition from the partially assembled SNARE complex to the fusion-competent SNAREpin. Thus, the results suggest that Syt1 and Ca2++ may drive SNARE zippering to facilitate membrane fusion.

Simultaneous AFM Force Spectroscopy and FRET Measurements on Single Biological Molecules

Department of Physics and Astronomy, Iowa State University, Ames, Iowa 50011. Abstract Single Molecule Fluorescence Resonance Energy Transfer (FRET) and single molecule force measurements with the Atomic Force Microscope (AFM) are two powerful techniques that have facilitated much progress in the biological sciences. However each of these techniques suffers from limitations that can be overcome by the use of a combined single molecule AFM-FRET approach. Here, we describe an instrument that successfully combines single molecule AFM with FRET to apply forces on individual biological molecules and simultaneously monitor their conformational dynamics. To validate this technique, we measured the force induced shearing of dye-labeled, double stranded DNA. Single DNA molecules were sheared and mechanical transitions corresponding to DNA rupture were correlated with changes in FRET.

Fret Measurements in Electrically Paced Adult Cardiac Myocytes Suggest the Phospholamban-SERCA2a Regulatory Complex is not Dissociated by Beat-To-Beat Elevations of Cytosolic Calcium

To investigate the regulatory interaction between cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a) and phospholamban (PLB), we expressed Cerulean-SERCA2a and YFP-PLB in adult rabbit cardiac muscle cells using adenovirus vectors. Confocal microscopy showed a fluorescence pattern of striations and longitudinal streaks indicating SERCA and PLB were correctly localized in the sarcoplasmic reticulum (SR). Bright perinuclear fluorescence was also observed. Fluorescence recovery after photobleaching (FRAP) experiments showed that SERCA and PLB were mobile over multiple sarcomeres on a timescale of tens of seconds. The concentration dependence of SERCA-PLB fluorescence resonance energy transfer (FRET) showed maximum FRET (FRETmax) was 30%, and also yielded the apparent dissociation constant (Kd). Addition of thapsigargin increased the apparent Kd, suggesting a reduced affinity of PLB for the pump in the presence of the inhibitor. Pacing of myocytes did not result in large changes in SERCA-PLB FRET, suggesting that the regulatory complex is not disrupted by beat-to-beat elevations of cytosolic calcium. The data are compatible with parallel experiments in heterologous cells indicating that the PLB-SERCA interaction is reduced, but not abolished by thapsigargin and calcium.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Detection of enzyme activity in orthotopic murine breast cancer by fluorescence lifetime imaging using a fluorescence resonance energy transfer–based molecular probe

Cancer-related enzyme activity can be detected noninvasively using activatable fluorescent molecular probes. In contrast to "always-on" fluorescent molecular probes, activatable probes are relatively nonfluorescent at the time of administration due to intramolecular fluorescence resonance energy transfer (FRET). Enzyme-mediated hydrolysis of peptide linkers results in reduced FRET and increase of fluorescence yield. Separation of signal from active and inactive probe can be difficult with conventional intensity-based fluorescence imaging. Fluorescence lifetime (FLT) measurement is an alternative method to detect changes in FRET. Thus, we investigate FLT imaging for in vivo detection of FRET-based molecular probe activation in an orthotopic breast cancer model. Indeed, the measured FLT of the enzyme-activatable molecular probe increases from 0.62 ns just after injection to 0.78 ns in tumor tissue after 4 h. A significant increase in FLT is not observed for an always-on targeted molecular probe with the same fluorescent reporter. These results show that FLT contrast is a powerful addition to preclinical imaging because it can report molecular activity in vivo due to changes in FRET. Fluorescence lifetime imaging exploits unique characteristics of fluorescent molecular probes that can be further translated into clinical applications, including noninvasive detection of cancer-related enzyme activity.

Metal-binding properties of Hpn from Helicobacter pylori and implications for the therapeutic activity of bismuth

Nickel is of particular importance to Helicobacter pylori in part because it acts as a cofactor of urease, which is critical to the survival of H. pylori. In this study the nickel storage, histidine-rich protein Hpn from H. pylori was converted into a Ni2+ probe by inserting it between two fluorescence resonance energy transfer (FRET) partners, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). The resulting construct, Hpn-FRET, exhibited a change in FRET upon the binding of Ni2+. Hpn-FRET has a moderate selectivity for Ni2+; it also responds to Zn2+ and Co2+ but not to other biometals. Competition experiments between Ni2+ and other metals plus the measured Kd values for Zn2+ and Ni2+ establish the selectivity order for Hpn-FRET as Zn2+ > Ni2+ > Co2+ ≫ other biometals. Bismuth is widely used as a therapeutic agent against H. pylori, and Hpn has been suggested as one of the possible targets. The dissociation constant of Bi3+ to Hpn-FRET was measured to be 6.19 × 10−5 M. Further experiments using Hpn-FRET in E. coli indicate that Hpn-FRET responds to Bi3+ but not to Ni2+ and Zn2+ inside E. coli. The result shows that unlike Ni2+ and Zn2+, which are tightly regulated in most bacteria, available Bi3+ can reach high micromolar levels inside E. coli.

Flow cytometric analysis of genetic FRET detectors containing variable substrate sequences

A genetic Fluorescence Resonance Energy Transfer (FRET) detector undergoes a post-translational modification (PTM)-induced conformational change that results in increased FRET. To test if the PTM-dependent FRET change can be quantified by flow cytometry, we purified and immobilized a genetic detector on microbeads and used flow cytometry to measure its FRET efficiency before and after Erk-2-mediated phosphorylation. The fluorescence ratio R between the acceptor and donor fluorescence, which was obtained by fitting a straight line through the data points in linear space, increases following phosphorylation, thus demonstrating that flow cytometry is capable of detecting a PTM-dependent FRET response. Furthermore, when Erk-2 and a genetic detector are coexpressed in bacteria, the measured R value changes with the substrate sequence with near single residue resolution. Similarly, the cells coexpressing the glycosylating enzyme O-GlcNAc transferase (OGT) and a genetic detector specific for OGT exhibit a PTM-induced change in FRET efficiency. Therefore, the combination of flow cytometry and a genetic detector may be useful to characterize the substrate specificity of a PTM enzyme and identify the sequences that are preferentially targeted for PTM in vivo.