Changes In Fluorescence Resonance Energy Transfer Research Articles

Apoptosis Detection in Retinal Ganglion Cells Using Quantitative Changes in Multichannel Fluorescence Colocalization.

KcapTR488 is a dual-fluorophore peptide sensor for the real-time reporting of programmed cell death by fluorescence imaging. KcapTR488 contains a nuclear localization sequence (NLS) conjugated with Texas Red, a caspase-cleavable sequence (DEVD), and a C-terminus conjugated to Alexa Fluor 488 (AF488). The synthesis and preliminary evaluation in cellulo of KcapTR488 for monitoring cell death by fluorescence imaging has been previously reported, but its utility in vivo has yet to be tested or validated. Herein, in vitro solution experiments verified the intramolecular fluorescence resonance energy transfer (FRET) between the two fluorophores and enabled a quantitative analysis of enzyme rates and selectivity. The sensor delivery kinetics in live rat models were quantified by ex vivo fluorescence microscopy. Studies in healthy control retinas demonstrated that KcapTR488 concentrated in the nucleus of retinal ganglion cells (RGC), with a strong colocalization of red and green fluorescence signals producing robust FRET signals, indicating an intact reporter. By contrast, using an acute but mild NMDA-induced retinal injury model, dual-color confocal ex vivo microscopy of cleaved KcapTR488 identified sensor activation as early as 2 h after injection. Quantitative changes in fluorescence colocalization were superior to changes in FRET for monitoring injury progression. Longitudinal monitoring revealed that the NLS-Texas Red fragment of the cleaved sensor moved out of the cell body, down the axon, and exited the retina, consistent with anterograde axonal transport. Thus, KcapTR488 may be a powerful tool to study RGC death pathways in live preclinical models of glaucoma.

DNA Origami Voltage Sensors for Transmembrane Potentials with Single-Molecule Sensitivity.

Signal transmission in neurons goes along with changes in the transmembrane potential. To report them, different approaches, including optical voltage-sensing dyes and genetically encoded voltage indicators, have evolved. Here, we present a DNA nanotechnology-based system and demonstrated its functionality on liposomes. Using DNA origami, we incorporated and optimized different properties such as membrane targeting and voltage sensing modularly. As a sensing unit, we used a hydrophobic red dye anchored to the membrane and an anionic green dye at the DNA to connect the nanostructure and the membrane dye anchor. Voltage-induced displacement of the anionic donor unit was read out by fluorescence resonance energy transfer (FRET) changes of single sensors attached to liposomes. A FRET change of ∼5% for ΔΨ = 100 mV was observed. The working mechanism of the sensor was rationalized by molecular dynamics simulations. Our approach holds potential for an application as nongenetically encoded membrane sensors.

Designing and Development of FRET-Based Nanosensor for Real Time Analysis of N-Acetyl-5-Neuraminic Acid in Living Cells

N-acetyl-5-neuraminic acid (NeuAc) plays crucial role in improving the growth, brain development, brain health maintenance, and immunity enhancement of infants. Commercially, it is used in the production of antiviral drugs, infant milk formulas, cosmetics, dietary supplements, and pharmaceutical products. Because of the rapidly increasing demand, metabolic engineering approach has attracted increasing attention for NeuAc biosynthesis. However, knowledge of metabolite flux in biosynthetic pathways is one of the major challenges in the practice of metabolic engineering. So, an understanding of the flux of NeuAc is needed to determine its cellular level at real time. The analysis of the flux can only be performed using a tool that has the capacity to measure metabolite level in cells without affecting other metabolic processes. A Fluorescence Resonance Energy Transfer (FRET)-based genetically-encoded nanosensor has been generated in this study to monitor the level of NeuAc in prokaryotic and eukaryotic cells. Sialic acid periplasmic binding protein (SiaP) from Haemophilus influenzae was exploited as a sensory element for the generation of nanosensor. The enhanced cyan fluorescent protein (ECFP) and Venus were used as Fluroscence Resonance Energy Transfer (FRET) pair. The nanosensor, which was termed fluorescent indicator protein for sialic acid (FLIP-SA), was successfully transformed into, and expressed in Escherichia coli BL21 (DE3) cells. The expressed protein of the nanosensor was isolated and purified. The purified nanosensor protein was characterized to assess the affinity, specificity, and stability in the pH range. The developed nanosensor exhibited FRET change after addition to NeuAc. The developed nanosensor was highly specific, exhibited pH stability, and detected NeuAc levels in the nanomolar to milimolar range. FLIP-SA was successfully introduced in bacterial and yeast cells and reported the real-time intracellular levels of NeuAc non-invasively. The FLIP-SA is an excellent tool for the metabolic flux analysis of the NeuAc biosynthetic pathway and, thus, may help unravel the regulatory mechanism of the metabolic pathway of NeuAc. Furthermore, FLIP-SA can be used for the high-throughput screening of E. coli mutant libraries for varied NeuAc production levels.

Detection of Streptavidin Based on Terminal Protection and Cationic Conjugated Polymer-Mediated Fluorescence Resonance Energy Transfer.

In this paper, a fast and simple strategy for sensitive detection of streptavidin (SA) was proposed based on terminal protection of small molecule-linked DNA and cationic conjugated polymer-mediated fluorescence resonance energy transfer (FRET). In principle, we designed a biotin-labelled DNA probe (P1) as the recognitive probe of SA, along with a complementary DNA probe (P2) to form double-stranded DNA (dsDNA) with P1. SYBR Green I (SG I) as a fluorescent dye was further used to specifically bind to dsDNA to emit stronger fluorescence. The cationic poly[(9,9-bis(6′-N,N,N-triethy-lammonium)hexyl) fluorenylene phenylene dibromide] (PFP) acted as the donor to participate in the FRET and transfer energy to the recipient SG I. In the absence of SA, P1 could not hybridize with P2 to form dsDNA and was digested by exonuclease I (Exo I); thus, only a weak FRET signal would be observed. In the presence of SA, biotin could specifically bind to SA, which protected P1 from Exo I cleavage. Then, P1 and P2 were hybridized into dsDNA. Therefore, the addition of SG I and PFP led to obvious FRET signal due to strong electrostatic interactions. Then, SA can be quantitatively detected by monitoring FRET changes. As the whole reagent reaction was carried out in 1.5 mL EP and detected in the colorimetric dish, the operation process of the detection system was relatively simple. The response time for each step was also relatively short. In this detection system, the linear equation was obtained for SA from 0.1 to 20 nM with a low detection limit of 0.068 nM (S/N = 3). In addition, this strategy has also achieved satisfactory results in the application of biological samples, which reveals the application prospect of this method in the future.

FRET-based nanosensors for monitoring and quantification of alcohols in living cells.

Odorants constitute a small and chemically diverse group of molecules with ethanol functioning as a key odorant that induces reproductive toxicity and adverse chronic effects on the liver. Analytical tools designed so far for the detection of odorant molecules are relatively invasive. Therefore, a tool that can measure the corresponding rate changes of ethanol concentration in real-time is highly desirable. Here in this work, we report a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor for in vivo quantification of ethanol at the cellular level with high spatial and temporal resolution. A human odorant-binding protein (hOBPIIa) was flanked by fluorescent proteins ECFP (Enhanced Cyan Fluorescent Protein) and Venus at the N- and C-terminus respectively. The constructed FRET nanosensor was named the fluorescent indicator protein for odorants (FLIPO). FLIPO allows in vitro and in vivo determination of FRET changes in a concentration-dependent manner. The developed nanosensor is highly specific to ethanol, stable to pH changes and provides rapid detection rate response. FLIPO-42 is the most efficient nanosensor created that measures ethanol with an apparent affinity (Kd) of 4.16 μM and covers the physiological range of 500 nM to 12 μM ethanol measurement. FLIPO-42 can measure ethanol dynamics in bacterial, yeast and mammalian cells non-invasively in real time which proves its efficacy as a sensing device in both prokaryotic and eukaryotic systems. Taken together, a prototype for a set of nanosensors was established, potentially enabling the monitoring of dynamic changes of ethanol and investigate its uptake and metabolism with subcellular resolution in vivo and ex vivo. Furthermore, the advent of a set of novel nanosensors will provide us with the tools for numerous medical, scientific, industrial and environmental applications which would help to illuminate their role in biological systems.

Red-Shifted FRET Biosensors for High-Throughput Fluorescence Lifetime Screening.

We have developed fluorescence resonance energy transfer (FRET) biosensors with red-shifted fluorescent proteins (FP), yielding improved characteristics for time-resolved (lifetime) fluorescence measurements. In comparison to biosensors with green and red FRET pairs (GFP/RFP), FPs that emit at longer wavelengths (orange and maroon, OFP/MFP) increased the FRET efficiency, dynamic range, and signal-to-background of high-throughput screening (HTS). OFP and MFP were fused to specific sites on the human cardiac calcium pump (SERCA2a) for detection of structural changes due to small-molecule effectors. When coupled with a recently improved HTS fluorescence lifetime microplate reader, this red-shifted FRET biosensor enabled high-precision nanosecond-resolved fluorescence decay measurements from microliter sample volumes at three minute read times per 1536-well-plate. Pilot screens with a library of small-molecules demonstrate that the OFP/MFP FRET sensor substantially improves HTS assay quality. These high-content FRET methods detect minute FRET changes with high precision, as needed to elucidate novel structural mechanisms from small-molecule or peptide regulators discovered through our ongoing HTS efforts. FRET sensors that emit at longer wavelengths are highly attractive to the FRET biosensor community for drug discovery and structural interrogation of new therapeutic targets.

Development of Novel Intramolecular FRET-Based ABC Transporter Biosensors to Identify New Substrates and Modulators.

Multidrug resistance protein 1 (MRP1) can efflux a wide variety of molecules including toxic chemicals, drugs, and their derivatives out of cells. Substrates of MRP1 include anti-cancer agents, antibiotics, anti-virals, anti-human immunodeficiency virus (HIV), and many other drugs. To identify novel substrates and modulators of MRP1 by exploiting intramolecular fluorescence resonance energy transfer (FRET), we genetically engineered six different two-color MRP1 proteins by changing green fluorescent protein (GFP) insertion sites, while keeping the red fluorescent protein (RFP) at the C-terminal of MRP1. Four of six recombinant proteins showed normal expression, localization, and transport activity. We quantified intramolecular FRET using ensemble fluorescence spectroscopy in response to binding of known substrate or ATP alone, substrate/ATP, and trapping of the transporter in closed conformation by vanadate. Recombinant MRP1 proteins GR-881, GR-888, and GR-905 exhibited reproducible and higher FRET changes under all tested conditions and are very promising for use as MRP1 biosensors. Furthermore, we used GR-881 to screen 40 novel anti-cancer drugs and identified 10 hits that potentially directly interact with MRP1 and could be substrates or modulators. Profiling of drug libraries for interaction with MRP1 can provide very useful information to improve the efficacy and reduce the toxicity of various therapies.

Detection of unique Ebola virus oligonucleotides using fluorescently-labeled phosphorodiamidate morpholino oligonucleotide probe pairs

Here we identify a low-cost diagnostic platform using fluorescently-labeled phosphorodiamidate morpholino oligonucleotide (PMO) probe pairs, which upon binding target oligonucleotides undergo fluorescence resonance energy transfer (FRET). Using a target oligonucleotide derived from the Ebola virus (EBOV), we have derivatized PMO probes with either Alexa Fluor488 (donor) or tetramethylrhodamine (acceptor). Upon EBOV target oligonulceotide binding, observed changes in FRET between PMO probe pairs permit a 25 pM lower limit of detection; there is no off-target binding within a complex mixture of nucleic acids and other biomolecules present in human saliva. Equivalent levels of FRET occur using PMO probe pairs for single or double stranded oligonucleotide targets. High-affinity binding is retained under low-ionic strength conditions that disrupt oligonucleotide secondary structures (e.g., stem-loop structures), ensuring reliable target detection. Under these low-ionic strength conditions, rates of PMO probe binding to target oligonucleotides are increased 3-fold relative to conventional high-ionic strength conditions used for nucleic acid hybridization, with half-maximal binding occurring within 10 min. Our results indicate an ability to use PMO probe pairs to detect clinically relevant levels of EBOV and other oligonucleotide targets in complex biological samples without the need for nucleic acid amplification, and open the possibility of population screening that includes assays for the genomic integration of DNA based copies of viral RNA.

Redistribution of SERCA calcium pump conformers during intracellular calcium signaling

The conformational changes of a calcium transport ATPase were investigated with molecular dynamics (MD) simulations as well as fluorescence resonance energy transfer (FRET) measurements to determine the significance of a discrete structural element for regulation of the conformational dynamics of the transport cycle. Previous MD simulations indicated that a loop in the cytosolic domain of the SERCA calcium transporter facilitates an open-to-closed structural transition. To investigate the significance of this structural element, we performed additional MD simulations and new biophysical measurements of SERCA structure and function. Rationally designed in silico mutations of three acidic residues of the loop decreased SERCA domain-domain contacts and increased domain-domain separation distances. Principal component analysis of MD simulations suggested decreased sampling of compact conformations upon N-loop mutagenesis. Deficits in headpiece structural dynamics were also detected by measuring intramolecular FRET of a Cer-YFP-SERCA construct (2-color SERCA). Compared with WT, the mutated 2-color SERCA shows a partial FRET response to calcium, whereas retaining full responsiveness to the inhibitor thapsigargin. Functional measurements showed that the mutated transporter still hydrolyzes ATP and transports calcium, but that maximal enzyme activity is reduced while maintaining similar calcium affinity. In live cells, calcium elevations resulted in concomitant FRET changes as the population of WT 2-color SERCA molecules redistributed among intermediates of the transport cycle. Our results provide novel insights on how the population of SERCA pumps responds to dynamic changes in intracellular calcium.

Enhanced Fluorescence Resonance Energy Transfer in G-Protein-Coupled Receptor Probes on Nanocoated Microscopy Coverslips

The G-protein-coupled receptor (GPCR) superfamily mediates cellular responses and communication across cellular membranes and is the largest known class of molecular targets with proven therapeutic value. For probing conformational changes of GPCRs and others in a live cell setting, fluorescence resonance energy transfer (FRET) is usually the method of choice. FRET probes often require careful labeling procedures, elaborate characterization, and assay optimization to provide both physiologically relevant probes with unaltered pharmacology and a sufficient dynamic range of the FRET changes. Here, we present an approach to optimize the energy transfer without changing the design of the FRET probe. We show that gold-coated glass coverslips reinforce the otherwise forbidden donor–acceptor energy transfer by virtual optimization of the dipole orientation. First, we confirm the resulting enhanced FRET efficiency on our nanocoatings for the inactive M1 muscarinic acetylcholine receptor (mAChR) labeled with a FRE...

‘Forbidden’ States Boost Fluorescence Resonance Energy Transfer (FRET) Studies of Membrane Receptors in Live Cells

For probing biomolecular interactions or conformational changes in a live-cell setting the distance depending Fluorescence resonance energy transfer (FRET) is often the method of choice. The G-protein-coupled receptor (GPCR) superfamily mediates cellular responses and communication across cellular membranes, and is the largest known class of molecular targets with proven therapeutic value. On the one hand, real-time monitoring of membrane receptors and their interactions is vital to understand how they act in concert. On the other hand, the design of GPCR FRET probes is crucial to ensure unhampered functionality and binding kinetics of the molecular complex. Thus, such FRET probes usually require labeling compromises concerning donor-acceptor positions, distances and orientations, which limit the detectability of emitter signals in general, and the dynamic range of the FRET changes in particular. Here, we present an approach to optimize the energy transfer without changing the design of the FRET probe. We show that gold coated glass cover slips allow reinforcing the otherwise forbidden donor-acceptor energy transfer by virtual optimization of the dipole orientation. First, we confirm the resulting enhanced FRET on our nano-coatings for the inactive M1 muscarinic acetylcholine receptors (mAChRs) labeled with a CFP-FlAsH pair in classical bleaching experiments. Second, we demonstrate the advantage of this enhanced FRET technique for ligand binding studies in live cells by the increased dynamic FRET response between the inactive and active state of the M1 mAChR receptor. We believe that our techniques has particular potential for pharmaceutical drug screening to boost non-ideal FRET probes, and thus amplify signal responses without interfering with the well-characterized molecular assay.

Dynamic Estimation of FRET Correction Factors to Study Redox Protein Interactions

Fluorescence resonance energy transfer (FRET) method is a key tool to determine molecular proximity and conformation changes of biomolecules in living cells. As the distance change between donor and acceptor fluorescent proteins attached to target proteins occurs in the range of 1-10 nm, an appreciable energy transfer can be detected at spatial and temporal resolution. Therefore it provides the most sensitive approach to study intercellular protein dynamics. Despite the earlier methods for characterization of FRET efficiency that accounts for donor and acceptor expression levels in cells; spectral bleed-through due to broad emission of fluorescent proteins results in inadequate computation of FRET, therefore efficiency and distance change cannot be determined accurately. Normalized FRET (nFRET) by using correction factors, were used to extract after FRET pair establishes an equilibrium state. However, photobleaching, varying expression of FRET pair, uncorrelated dipole orientations of fluorescent proteins, heterogeneous distance changes of linkers between donor and acceptor result in incomplete computation of nFRET since correction factors were not dynamically determined during data acquisition. Thus, the use of single bleed through coefficient for computation of netFRET values may also results in overestimation of efficiency and distance. We demonstrated that donor and acceptor bleed-through values changes during the data acquisition. Therefore, netFRET cannot be computed by a single donor-acceptor correction factors. Alternatively, we presented here dynamic coefficient estimation method to determine correction factors by using the ratio of emissions from FRET channel and donor/acceptor channels for each frame after fitting pixel intensity values and later used them to compute nFRET efficiencies. The method yields a 0.38 nFRET values for Cerulean-Venus (Cer17Ven) FRET pair separated by a short linker, however the use of static coefficients of average or minimum factors yields 0.34 nFRET value. Single coefficient incorrectly reports distance change when the measurements were taken before an equilibrium state. Furthermore, method was used to measure heme induced intercellular conformation changes of Cytochrome c labeled with Cer and Ven fluorescent pairs. The distance after Cyt c folded by Heme Lyase was accurately computed. As a result, a new approach was demonstrated to determine the nFRET efficiency and its direct use to compute distance changes. Taking all values during the data acquisition eliminated the coefficient dependent variation.

Exploring Drug-Receptor Interaction Kinetics: Lessons from a Sigma-1 Receptor Transmembrane Biosensor.

An important field of study in pharmacology comprises the investigation of drug-target interaction kinetics. Thus, assessing both the lifetime of a drug on its receptor (i.e., drug-target residence time; Copeland, 2016) and the magnitude of drug-mediated receptor activation (i.e., drug efficacy) across the time are critical to understand in vivo pharmacological activity of small-molecule drugs. Of note, while classical in vitro methods view drug-receptor interaction in terms of equilibrium affinity, the residence time model considers the dynamics of receptor conformational rearrangements, which affect drug association and dissociation. Although, classical binding experiments can also address kinetics questions, they are tedious and very time consuming. Accordingly, monitoring drug-receptor interaction dynamics by means of receptor biosensors has become fundamental for understanding how drugs trigger receptor activity over the time. Precisely, in the last years, a number of Fluorescence Resonance Energy Transfer (FRET)-based assays have been developed to accurately display drug-receptor interaction in real time (Lohse et al., 2012). Indeed, one of the most outstanding methods consists of assessing intramolecular conformational rearrangements upon receptor challenge by monitoring intramolecular FRET changes (Vilardaga et al., 2009). Thus, a FRET-based receptor biosensor is built by fusing both donor and acceptor fluorophores to the receptor sequence (Vilardaga et al., 2009). Importantly, a general consensus has prompted to basically attach these molecules (i.e., cyan and yellow fluorescent proteins, CFP and YFP, respectively) intracellularly, this is, in the cytosolic side of the receptor's structure (Figure ​(Figure1A).1A). Accordingly, when the receptor is activated and a conformational rearrangement occurs the distance and/or orientation of the fluorophores within the receptor biosensor changes and it is possible to monitor FRET changes in real time, thus permitting to finely characterize receptor's activation. Needless to say, although precision is higher than that obtained in classical binding assays, the present biosensors cannot discern between receptors expressed at the cell surface or intracellularly, thus much effort is needed in order to exactly elucidate ligand-receptor kinetics constants.

Tracking the Activity of mTORC1 in Living Cells Using Genetically Encoded FRET-based Biosensor TORCAR.

Mechanistic target of rapamycin complex 1 (mTORC1) is a highly conserved serine/threonine protein kinase that responds to multiple distinct signals (e.g., growth factors, amino acids, stress, and energy level) and coordinates cell growth and proliferation. The underlying molecular mechanisms by which these stimuli regulate the activity of mTORC1 are still not fully understood. The spatial compartmentalization of mTORC1 signaling has been suggested as an important mechanism for mTORC1 to achieve the signal specificity and efficiency. To examine the spatial regulation of the activity of mTORC1 in live cells, we describe a protocol using a newly developed molecular tool, a genetically encoded fluorescence resonance energy transfer (FRET)-based mTORC1 activity reporter, TORCAR. When expressed in the cell, TORCAR acts as a surrogate substrate of mTORC1, and exhibits a change in FRET in response to phosphorylation by mTORC1. Genetically targeting TORCAR to specific subcellular locations further allows for the characterization of spatial compartmentalized mTORC1 signaling. © 2016 by John Wiley & Sons, Inc.

A Guide to Fluorescent Protein FRET Pairs.

Förster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies.

A Small Loop in the SERCA N-Domain Facilitates the Transition to a Compact Conformation

The cardiac sarco/endoplasmic reticulum (SR) calcium ATPase (SERCA) and its inhibitor phospholamban (PLB) are key regulators of calcium levels in cardiac cells. SERCA contributes to calcium re-uptake from the cytosol into the SR, resulting in the relaxation of the muscles. PLB phosphorylation leads to SERCA relief-of-inhibition, and subsequently improved contractility and faster relaxation due to increased SR calcium load. The current project is aimed at revealing the details of the SERCA transition from an open to a closed conformation. In addition, the experiments provide the insight into the mechanism of the SERCA relief of inhibition after PLB phosphorylation. Molecular dynamics (MD) simulations led us to hypothesize that H-bonds between a small loop in SERCA N-domain and the A-domain facilitate SERCA headpiece closure. We propose that the cytoplasmic domain of phosphorylated PLB facilitates the transition to a closed conformation by interaction with this discrete loop of SERCA N-domain. Other groups had reported that mutation of N-loop residues reduces ATPase activity by 35-60%. Using fluorescence resonance energy transfer (FRET) experiments and MD simulations, we find that SERCA N-loop mutations impair the closure of cytoplasmic headpiece. Furthermore, SERCA precipitation with GST-PLB is reduced upon N-loop mutation. MD simulations of a SERCA-PLB docking model suggest that PLB phosphorylation facilitates an interaction of the PLB cytoplasmic domain with SERCA A-domain and the N-domain loop, leading to a more compact conformation of the SERCA headpiece. Indeed, we observe a change in FRET between SERCA and PLB after PLB phosphorylation, consistent with a conformational change of the SERCA-PLB complex. Together, our data implicate a small N-domain loop in SERCA transitioning to a compact conformation. This discrete loop might potentially become a new target for rational design of novel therapeutics to modulate SERCA activity.

Conjugated Polythiophene/Porphyrin Complex for Rapid and Simple Detection of Bacteria in Drinking Water

An electrostatic complex combining anionic water‐soluble conjugated polythiophene (PTP) with commercial cationic porphyrin ([5,10,15,20‐tetrakis(4‐(trimethylammonio)‐phenyl)‐21H,23H‐porphine tetratosylate] (TMP)) has been designed for rapid and simple detection of bacteria infection in drinking water by applying fluorescence resonance energy transfer (FRET) strategy. There is efficient FRET from PTP to TMP for the electrostatic complex of PTP/TMP. Upon the addition of bacteria, the cationic TMP is adsorbed tightly to the negatively charged membrane surface of bacteria through electrostatic interactions, making the PTP reside in much farther proximity to TMP than that in the absence of bacteria. Therefore, much weaker FRET is observed. Interesting, the detection for bacteria based on FRET changes of PTP/TMP complex is visual in view of the color changes under ultraviolet (UV) irradiation. This assay takes advantage of the light‐harvesting property of conjugated polymer and the ratiometric measurements for minimizing the nonspecific binding effect. The strategy is promising in pathogen detection in drinking water and other sources. image

Abstracts

Abstracts

Rapid structural change in synaptosomal-associated protein 25 (SNAP25) precedes the fusion of single vesicles with the plasma membrane in live chromaffin cells

The SNARE complex consists of the three proteins synaptobrevin-2, syntaxin, and synaptosomal-associated protein 25 (SNAP25) and is thought to execute a large conformational change as it drives membrane fusion and exocytosis. The relation between changes in the SNARE complex and fusion pore opening is, however, still unknown. We report here a direct measurement relating a change in the SNARE complex to vesicle fusion on the millisecond time scale. In individual chromaffin cells, we tracked conformational changes in SNAP25 by total internal reflection fluorescence resonance energy transfer (FRET) microscopy while exocytotic catecholamine release from single vesicles was simultaneously recorded using a microfabricated electrochemical detector array. A local rapid and transient FRET change occurred precisely where individual vesicles released catecholamine. To overcome the low time resolution of the imaging frames needed to collect sufficient signal intensity, a method named event correlation microscopy was developed, which revealed that the FRET change was abrupt and preceded the opening of an exocytotic fusion pore by ∼90 ms. The FRET change correlated temporally with the opening of the fusion pore and not with its dilation.

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.