Synthesis and characterization of improved chloride-sensitive fluorescent indicators for biological applications

A class of N-substituted quinoline compounds has been introduced recently for the fluorescence measurement of Cl concentration in biological preparations. The most Cl-sensitive compound was 6-methoxy-N-[3-sulfopropyl] quinolinium with peak excitation and emission wavelengths of 350 and 442 nm and a Stern-Volmer constant for quenching by Cl of 118 M-1. Six water-soluble quinoline derivatives were synthesized and characterized for the purposes of increasing Cl sensitivity, adding ester functions for cell trapping, and red-shifting the fluorescence peak wavelengths. Acetic acid ester functions were added at the N-, 2-, and 6-positions of the quinoline ring. The best ester compound, N-(6-methoxyquinolyl)acetoethyl ester (MQAE), was water soluble (270 g/liter at 23 degrees C; octanol:H2O partition coefficient of 0.009), had a high Cl sensitivity (Stern-Volmer constant 200 M-1), peak excitation and emission wavelengths of 355 and 460 nm, a fluorescence lifetime of 21.6 ns, and a molar absorbance of 4850 M-1 cm-1 (320 nm). MQAE fluorescence was not altered by the physiological anions HCO3, SO4, and PO4, by cations, or by pH. MQAE was used to measure chloride transport in liposome membranes and in cultured LLC-PK1 cells in monolayer; MQAE leaked out of cells less than 20% in 60 min at 37 degrees C. The physical, optical, and anion quenching properties for the series of ester compounds were determined to establish a set of structure-activity correlates.     

Membrane chloride transport measured using a chloride-sensitive fluorescent probe

Transport of chloride across cell membranes through exchange, cotransport, or conductive pathways is a subject of great biological importance. Current methods of measurement are restricted in their sensitivity, time resolution, and applicability. A new transport measurement technique has been developed on the basis of the fluorescence quenching by chloride of the dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). SPQ fluorescence quenching by chloride is rapid (less than 1 ms) and sensitive, with a greater than 50% decrease in fluorescence at 10 mM chloride. SPQ fluorescence is not altered by other physiological anions or by pH and can be used to measure both neutral and conductive transport processes. The high water solubility and membrane permeability properties of SPQ make it ideal for use in both membrane vesicles and cells. Chloride transport determined with SPQ was validated by measurement of erythrocyte chloride/anion exchange and membrane vesicle chloride conductance.     

Chloride-sensitive fluorescent indicators

Three fluorescent halide-sensitive quinolinium dyes have been produced by the reaction of the 6-methylquinoline heterocyclic nitrogen base with methyl bromide, methyl iodide, and 3-bromo-1-propanol. The quaternary salts, unlike the precursor molecule, are readily water soluble and the fluorescence intensity of these salts is reduced in the presence of aqueous chloride, bromide, and iodide ions, allowing halide solution concentrations to be determined using well-known Stern-Volmer kinetics. One of the dyes, dye 1, has a chloride Stern-Volmer constant of 255 mol(-1) dm(3) which is more than twice that of SPQ [6-methoxy-N-(3-sulfopropyl)quinolinium] used in recent physiological measurements to measure intracellular chloride levels. The dyes have been characterized using steady-state fluorescence spectroscopy and are compared to three similar dyes based on the 6-methoxyquinoline nucleus, reported earlier by the authors, and also to dyes reported by Krapf et al. (Anal. Biochem. 169, 142-150, 1988). The interference of aqueous anions and the potential for using these dyes in biological halide-sensing applications are discussed.     

Characterisation of chloride transport at the tonoplast of higher plants using a chloride-sensitive fluorescent probe

The characteristics of Cl^– transport in isolated tonoplast vesicles from red-beet (Beta vulgaris L.) storage tissue have been investigated using the Cl^–-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)-quinolinium (SPQ). The imposition of (inside) positive diffusion potentials, generated with K⁺ and valinomycin, increased the initial rate of Cl^– transport, demonstrating that Cl^– could be electrically driven into the vesicles. Chloride influx was unaffected by SO ₄ ^2- , but was competitively blocked by NO ₃ ^– , indicating that both Cl^– and NO ₃ ^– may be transported by the same porter. In some preparations, increases in free-Ca²⁺ concentration from 10^–8 to 10^–5 mol·dm^–3 caused a significant decrease in Cl^– influx, which may indicate that cytosolic Ca²⁺ concentration has a role in controlling Cl^– fluxes at the tonoplast. However, this effect was only seen in about 50% of membrane preparations and some doubt remains over its physiological significance. A range of compounds known to block anion transport in other systems was tested, and some partially blocked Cl^– transport. However, many of these inhibitors interfered with SPQ fluorescence and so only irreversible effects could be tested. The results are discussed in the context of recent advances made using the patch-clamp technique on isolated vacuoles.Abbreviations and Symbols BTP 1,3-bis[tris(hydroxymethyl)-methylamino]propane - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - membrane potential - pH pH gradient - SPQ 6-methoxy-1-(3-sulfonatopropyl)quinolinium - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine     

The use of a chloride-sensitive fluorescent probe to measure chloride transport in isolated tonoplast vesicles

A fluorescence method for the direct measurement of Cl^- transport in isolated tonoplast vesicles is described. This technique utilises the Cl^--sensitive fluorescent compound, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ). This is a water-soluble compound with excitation and emission wavelengths of 350 and 440 nm, respectively. Its fluorescence is quenched by Cl^-, Br^-, I^-, SCN^-, NO ₂ ^- and tetraphenylborate but not by NO ₃ ^- , SO ₄ ^2- , iminodiacetate or malate. These effects are independent of pH. This compound was loaded into tonoplast vesicles from red beet (Beta vulgaris L.) storage roots or from barley (Hordeum vulgare L.) roots by incubation at 37° C and the external probe was then removed by repeated centrifugation of the vesicles in SPQ-free medium. In this way a large proportion of the observed fluorescence signal was from the interior of the vesicles, and its quenching could be used to monitor, quantitatively, and in real time, the intravesicular Cl^- concentration. In this paper we describe some of the problems encountered in using this probe to measure Cl^- transport in tonoplast vesicles, how these were overcome and some characteristics of Cl^- transport at the tonoplast as measured by the probe.Abbreviations and symbols BTP 1,3-bis[tris(hydroxymethyl)-methylamino-propane - DTT dithiothreitol - membrane potential - pH pH gradient - PPase inorganic pyrophosphatase - PPi inorganic pyrophosphate - SPQ 6-methoxy-1-(3-sulfonatopropyl)quinolinium - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Quenching of superoxide radicals by green fluorescent protein

Green fluorescent proteins (GFP) are widely used in vivo molecular markers. These proteins are particularly resistant, and maintain function, under a variety of cellular conditions such as pH extremes and elevated temperatures. Green fluorescent proteins are also abundant in several groups of marine invertebrates including reef-forming corals. While molecular oxygen is required for the post-translational maturation of the protein, mature GFPs are found in corals where hyperoxia and reactive oxygen species (ROS) occur due to the photosynthetic activity of algal symbionts. In vitro spin trapping electron paramagnetic resonance and spectrophotometric assays of superoxide dismutase (SOD)-like enzyme activity show that wild type GFP from the hydromedusa, Aequorea victoria, quenches superoxide radicals (O2*-)) and exhibits SOD-like activity by competing with cytochrome c for reaction with O2*-. When exposed to high amounts of O2*- the SOD-like activity and protein structure of GFP are altered without significant changes to the fluorescent properties of the protein. Because of the distribution of fluorescent proteins in both the epithelial and gastrodermal cells of reef-forming corals we propose that GFP, and possibly other fluorescent proteins, can provide supplementary antioxidant protection.     

Measuring calcium signaling using genetically targetable fluorescent indicators

Genetically encoded Ca2+ indicators allow researchers to quantitatively measure Ca2+ dynamics in a variety of experimental systems. This protocol summarizes the indicators that are available, and highlights those that are most appropriate for a number of experimental conditions, such as measuring Ca2+ in specific organelles and localizations in mammalian tissue-culture cells. The protocol itself focuses on the use of a cameleon, which is a fluorescence resonance-energy transfer (FRET)-based indicator comprising two fluorescent proteins and two Ca2+-responsive elements (a variant of calmodulin (CaM) and a CaM-binding peptide). This protocol details how to set up and conduct a Ca2+-imaging experiment, accomplish offline data processing (such as background correction) and convert the observed FRET ratio changes to Ca2+ concentrations. Additionally, we highlight some of the challenges in observing organellar Ca2+ and the alternative strategies researchers can employ for effectively calibrating the genetically encoded Ca2+ indicators in these locations. Setting up and conducting an initial calibration of the microscope system is estimated to take approximately 1 week, assuming that all the component parts are readily available. Cell culture and transfection is estimated to take approximately 3 d (from the time of plating cells on imaging dishes). An experiment and calibration will probably take a few hours. Finally, the offline data workup can take approximately 1 d depending on the extent of analysis.     

Genetically encoded fluorescent indicators for live cell pH imaging

Background

Intracellular pH underlies most cellular processes. There is emerging evidence of a pH-signaling role in plant cells and microorganisms. Dysregulation of pH is associated with human diseases, such as cancer and Alzheimer's disease.

A construct with fluorescent indicators for conditional expression of miRNA

Background  

Transgenic RNAi holds promise as a simple, low-cost, and fast method for reverse genetics in mammals. It may be particularly useful for producing animal models for hypomorphic gene function. Inducible RNAi that permits spatially and temporally controllable gene silencing in vivo will enhance the power of transgenic RNAi approach. Furthermore, because microRNA (miRNA) targeting specific genes can be expressed simultaneously with protein coding genes, incorporation of fluorescent marker proteins can simplify the screening and analysis of transgenic RNAi animals.     

Effects of photolabile calcium chelators on fluorescent calcium indicators.

The fluorescence properties of the calcium indicators Fura-2 and Fluo-3 have been investigated in the presence of the 'caged calcium' photolabile chelators Nitr-5 and DM-nitrophen. The excitation spectra of dilute solutions of these indicators was distorted by the presence of photolabile chelators, owing to differential absorbance of excitation light by the chelators, as well as calcium-dependent fluorescence of the chelators themselves. This distortion was altered on partial photolysis of the chelators, due to changes in their absorbance and fluorescence. At high concentrations of indicators (100 microM) and photolabile chelators (10 mM), similar to those used experimentally, DM-nitrophen quenched the fluorescence of Fluo-3 at low calcium concentrations. The results suggest that Fura-2 may be used with either chelator, and Fluo-3 with Nitr-5, to measure calcium released on photolysis of the caged compounds, but that careful calibration of the chelator-indicator mixture after the appropriate degree of photolysis is necessary.     

Dendrimer-based fluorescent indicators: in vitro and in vivo applications

BackgroundThe development of fluorescent proteins and synthetic molecules whose fluorescence properties are controlled by the environment makes it possible to monitor physiological and pathological events in living systems with minimal perturbation. A large number of small organic dyes are available and routinely used to measure biologically relevant parameters. Unfortunately their application is hindered by a number of limitations stemming from the use of these small molecules in the biological environment.ConclusionWe believe that the proposed architecture can represent a useful and novel tool in fluorescence imaging that can be widely applied in conjunction with a broad range of sensing dyes and experimental setups.     

Near-membrane iminocoumarin-based low affinity fluorescent Ca(2+) indicators

Two new potential near-membrane iminocoumarin-based fluorescent Ca(2+) indicators were synthesized and the spectral profiles of their free and Ca(2+) bound forms were studied. The probes incorporate in their BAPTA-related structures, the 3-(benzimidazolyl)iminocoumarin or the 3-(benzothiazolyl)iminocoumarin moiety, substituted at the imino nitrogen with an n-dodecyl lipophilic chain. The compounds are excited with visible light and have Ca(2+) dissociation constant values of 5.50 and 4.49 microM, respectively, the highest reported to date in the literature. Fluorescence spectra studies indicated a clear shift in their excitation wavelength maxima upon Ca(2+) binding along with changes in fluorescence intensity that enable the compounds to be used as ratiometric near-membrane, low Ca(2+) affinity probes.     

Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators

Current methods for determining ambient redox potential in cells are labor-intensive and generally require destruction of tissue. This precludes single cell or real time studies of changes in redox poise that result from metabolic processes or environmental influences. By substitution of surface-exposed residues on the Aequorea victoria green fluorescent protein (GFP) with cysteines in appropriate positions to form disulfide bonds, reduction-oxidation-sensitive GFPs (roGFPs) have been created. roGFPs have two fluorescence excitation maxima at about 400 and 490 nm and display rapid and reversible ratiometric changes in fluorescence in response to changes in ambient redox potential in vitro and in vivo. Crystal structure analyses of reduced and oxidized crystals of roGFP2 at 2.0- and 1.9-A resolution, respectively, reveal in the oxidized state a highly strained disulfide and localized main chain structural changes that presumably account for the state-dependent spectral changes. roGFP1 has been targeted to the mitochondria in HeLa cells. Fluorometric measurements on these cells using a fluorescence microscope or in cell suspension using a fluorometer reveal that the roGFP1 probe is in dynamic equilibrium with the mitochondrial redox status and responds to membrane-permeable reductants and oxidants. The roGFP1 probe reports that the matrix space in HeLa cell mitochondria is highly reducing, with a midpoint potential near -360 mV (assuming mitochondrial pH approximately 8.0 at 37 degrees C). In other work (C. T. Dooley, T. M. Dore, G. Hanson, W. C. Jackson, S. J. Remington, and R. Y. Tsien, submitted for publication), it is shown that the cytosol of HeLa cells is also unusually reducing but somewhat less so than the mitochondrial matrix.     

Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo

One of the challenges for modern neuroscience is to understand the rules of concerted neuronal function in vivo. This question can be addressed using noninvasive high-resolution imaging techniques like two-photon microscopy. This protocol describes a versatile approach for in vivo two-photon calcium imaging of neural networks, stained with membrane-permeant fluorescent-indicator dyes. It is based on a targeted pressure ejection of the dye into the tissue of interest and can be used for a large spectrum of indicator dyes, including Oregon Green 488 BAPTA-1 acetoxymethyl ester and Fura-2 acetoxymethyl ester. Through the use of dye mixtures and multicolor imaging, this technique allows the visualization of distinct neurons and glial cells up to 500 microm below the brain surface. It is suitable for staining the brain tissue of various different species (e.g., mouse, rat, cat and zebrafish) at all developmental stages. When combined with brain microendoscopy, it allows the monitoring of intracellular calcium signals in awake, behaving animals. The total time required to carry out the protocol, including dissection and cell staining, is approximately 2 h. Thereafter, imaging experiments might be performed for at least 6 h.     

Interactions between inositol trisphosphate receptors and fluorescent Ca2+ indicators.

Fura-2 and BAPTA were previously shown to be competitive antagonists of inositol trisphosphate (InsP3) receptors, but for practical reasons the analyses were performed at pH 8.3. We recently developed a scintillation proximity assay (SPA) for pure cerebellar InsP3 receptors which allows low affinity interactions to be characterized and is readily applicable to scarce or expensive ligands. In the present study, we use SPA to demonstrate that at pH 7.2, many of the commonly used fluorescent Ca2+ indicators reversibly displace 3H-InsP3 from its receptor and that they differ substantially in their affinities for the InsP3 receptor (IC50 = 6.5-137 microM). Recombinant type 1 InsP3 receptors expressed in Sf9 cells were used to examine 3H-InsP3 binding in cytosol-like medium: both fura-2 (IC50 = 796 +/- 86 microM) and Ca Green-5N (IC50 = 62 +/- 7 microM) completely inhibited the binding, but only in their Ca(2+)-free forms. Similar results were obtained with type 3 InsP3 receptors. We conclude that many Ca2+ indicators in their Ca(2+)-free forms compete with InsP3 for binding to its receptor, and that for Ca Green-5N the interaction occurs with sufficient affinity to significantly perturb physiological responses.     

Imaging local estrogen production in single living cells with recombinant fluorescent indicators

Estrogens are steroid hormones with many systemic effects in addition to development and maintenance of the female reproductive system, and ligands of estrogen receptors are of clinical importance because of their use as oral contraceptive, hormone replacement and antitumoral therapy. In addition, tumoral tissues have been found to express aromatase and other steroidogenic enzymes synthesizing estradiol. To aid in the understanding of these processes, we have developed assays to image the local production of estrogens in isolated living mammalian cells. We constructed biosensors based on estrogen receptor α ligand binding domain and fluorescent proteins by following two approaches. First, the ligand binding domain and a short fragment of steroid receptor coactivator-1 were appended to a circularly permuted yellow fluorescent protein to construct an excitation ratio estrogen indicator. In the second strategy, we constructed emission ratio sensors based on fluorescence resonance energy transfer, containing the ligand binding domain flanked by donor and acceptor fluorescent proteins. Estrogens altered the fluorescence signal of cells transfected with the indicators in a dose-dependent manner. We imaged local estrogen production in adrenocortical H295 cells expressing aromatase and transfected with the fluorescent sensors. In addition, paracrine detection was observed in HeLa cells harboring the indicators and co-cultured with H295 cells. This imaging approach may allow detection of physiological levels of these hormones in suitable animal models.     

Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators

Changes in the redox equilibrium of cells influence a host of cell functions. Alterations in the redox equilibrium are precipitated by changing either the glutathione/glutathione-disulfide ratio (GSH/GSSG) and/or the reduced/oxidized thioredoxin ratio. Redox-sensitive green fluorescent proteins (GFP) allow real time visualization of the oxidation state of the indicator. Ratios of fluorescence from excitation at 400 and 490 nm indicate the extent of oxidation and thus the redox potential while canceling out the amount of indicator and the absolute optical sensitivity. Because the indicator is genetically encoded, it can be targeted to specific proteins or organelles of interest and expressed in a wide variety of cells and organisms. We evaluated roGFP1 (GFP with mutations C48S, S147C, and Q204C) and roGFP2 (the same plus S65T) with physiologically or toxicologically relevant oxidants both in vitro and in living mammalian cells. Furthermore, we investigated the response of the redox probes under physiological redox changes during superoxide bursts in macrophage cells, hyperoxic and hypoxic conditions, and in responses to H(2)O(2)-stimulating agents, e.g. epidermal growth factor and lysophosphatidic acid.     

Application of fluorescent indicators to analyse intracellular calcium and morphology in filamentous fungi

A novel staining and quantification method to investigate changes in intracellular calcium levels [Ca²⁺]_i and morphology in filamentous fungus is presented. Using a simple protocol, two fluorescent dyes, Fluo-4-AM and Cell trace calcein red-orange-AM were loaded into the filamentous fungus Penicillium chrysogenum. The present study investigates the applicability of using Ca²⁺-sensitive dye to quantify and image [Ca²⁺]_i in P. chrysogenum cultures chosen for its potential as an experimental system to study Ca²⁺ signalling in elicited cultures. The dye loading was optimised and investigated at different pH loading conditions. It was observed that the fluorophore was taken up throughout the hyphae, retaining cell membrane integrity and no dye compartmentalisation within organelles was observed. From the fluorescent plate-reader studies a significant rise (p < 0.001) in the relative fluorescence levels corresponding to [Ca²⁺]_i levels in the hyphae was observed when challenged with an elicitor (mannan oligosaccharide, 150 mg L^?1) which was dependent upon extracellular calcium. Concurrently a novel application of dye-loaded hyphae for morphological analysis was also examined using the imaging software Filament Tracer (Bitplane). Essential quantitative mycelial information including the length and diameter of the segments and number of branch points was obtained using this application based on the three-dimensional data.