Styryl Dye Research Articles

Novel, N-ethyl-2-styrylquinolinum iodides as fluorophores for monitoring of polymerization process, Part I

A series of of p-substituted 2-styrylquinolinium iodides were prepared by the condensation of N-ethyl-2-methylquinolinium salts with p-substituted benzaldehydes. The spectroscopic properties of the styryl quinolinium dyes are characterized in organic solvents of varying polarities. The electronic absorption and fluorescence emission spectra of the dyes demonstrate their high sensitivity to the nature of substituents introduced into the aromatic ring. The dyes were investigated as fluorescent probes for monitoring the progress of the photochemically initiated free-radical polymerization of a mixture of 2-ethyl-2-(hydroxymethyl)-1,3-propanediol triacrylate and 1-methyl-2-pyrrolidinone. During the course of the polymerization an increase in the fluorescence intensity of the dyes by at least one order of magnitude was recorded; a feature which renders the dyes as good fluorescent probes for such polymerization reactions. The term “probe sensitivity” has been defined and appears in the range from 0.08 to 11 for the styryl dyes.

Investigation Of Electrogenic Partial Reactions In Detergent-solubilized Na,K-ATPase

Electrochromic styryl dyes are in use now for almost two decades to detect ion movements in various P-type ATPases. The extremely hydrophobic dye molecules have a high partition coefficient in favor of the hydrophobic core of lipid phase of membrane preparations. Fluorescence changes are obtained by modification of local electric fields in the membrane dielectric produced by ions bound to or released from binding sites of the ion pumps. To obtain significant signals a prerequisite is a high density of active proteins in the membranes. This limitation could be overcome by solubilization of the Na,K-ATPase in mixed micelles of protein/lipid/detergent obtained by incubation of microsomal membranes from rabbit kidney with dodecyl maltoside. In this preparation the specific enzyme activity of the Na,K-ATPase was reduced compared to that in native membranes. This effect was assigned to the highly affected lipid environment of the singularized proteins which may be depleted of specific lipid components and the content of dodecyl maltoside. The fluorescence changes which were detected with the styryl dye RH421 showed smaller amplitudes than in the case of purified membrane preparations, however, the responses on Na+ binding, Na+ release upon enzyme phosphorylation and conformation transition, and subsequent K+ binding in the E2P conformation were clearly detectable. Na+ binding affinity and its dependence on Mg2+ concentration and buffer pH, as well as K+ affinity were comparable to the results obtained with native preparations. The transfer of the method to solubilized ion pumps will allow investigations of mutants isolated from cell in which they were expressed in low density. In addition, this approach will possibly extend method also to the investigation of ion channel molecules by electrochromic styryl dyes.

New Dyes With Fast Voltage-Dependent Changes In Membrane SHG

Laser scanning second harmonic generation (SHG) microscopy has shown significant promise for membrane potential imaging with voltage sensitive dyes (VSDs), possessing significant advantages over fluorescence-based imaging modalities. Through simultaneous patch-clamping and non-linear imaging of cells, SHG has been found to exhibit sensitivities to trans-membrane potential that are up to four times better than those obtained under optimal conditions using one-photon fluorescence imaging (Millard et al., 2004). For styryl dyes, while electrochromism is the dominating photophysical mechanism of fluorescence, some ANEP-based dyes display slow SHG voltage responses, suggesting that chromophore membrane reorientation or redistribution may be involved. The mechanism of the SHG response is not entirely understood and necessitates additional study in order to fully optimize this imaging modality. We report on our further investigation of the time dependence of the voltage sensitivity of SHG and simultaneous two-photon fluorescence imaging, using “fast” voltage-switching experiments. The response kinetics of resonance enhanced SHG from several styryl dyes developed in our laboratory, including di-3-ANEPPDHQ, as well as di-4-ANEPPTEA and a new fluorinated derivative, have been determined. Voltage-clamped neuroblastoma cells stained with these dyes were imaged with 1064 nm excitation from a mode locked fiber-based laser source. For SHG, these VSDs were found to exhibit moderately large voltage sensitivities in addition to fast kinetic responses. Our results suggest that voltage sensitive dyes can be developed which have both large SHG signal changes and the requisite speed for use as a practical tool for measuring electrical activity in neuronal systems.(Supported by NIH grant EB001963).

Two Pathways of Synaptic Vesicle Retrieval Revealed by Single-Vesicle Imaging

Synaptic vesicle recycling is essential for maintaining efficient synaptic transmission. Detailed dissection of single-vesicle recycling still remains a major challenge. We have developed a fluorescent pH reporter that permits us to follow the fate of individual vesicles at hippocampal synapses after exocytosis. Here we show that, during low-frequency stimulation, single-vesicle fusion leads to two distinct vesicle internalizations, instead of one, as in general perception: one by a fast endocytosis pathway ( approximately 3 s), the other by a slow endocytosis pathway (after 10 s). The exocytosed vesicular proteins are preferentially recaptured in both pathways. RNAi knockdown of clathrin inhibits both pathways. As stimulation frequency increases, the number of endocytosed vesicles begins to match antecedent exocytosis. Meanwhile, the slow endocytosis is accelerated and becomes the predominant pathway. These results reveal that two pathways of endocytosis are orchestrated during neuronal activity, establishing a highly efficient endocytosis at central synapses.

Caveolin-2 is required for apical lipid trafficking and suppresses basolateral recycling defects in the intestine of Caenorhabditis elegans.

Caveolins are plasma membrane-associated proteins that colocalize with, and stabilize caveolae. Their functions remain unclear although they are known to be involved in specific events in cell signaling and endocytosis. Caenorhabditis elegans encodes two caveolin genes, cav-1 and cav-2. We show that cav-2 is expressed in the intestine where it is localized to the apical membrane and in intracellular bodies. Using the styryl dye FM4-64 and BODIPY-labeled lactosylceramide, we show that the intestinal cells of cav-2 animals are defective in the apical uptake of lipid markers. These results suggest parallels with the function of caveolins in lipid homeostasis in mammals. We also show that CAV-2 depletion suppresses the abnormal accumulation of vacuoles that result from defective basolateral recycling in rme-1 and rab-10 mutants. Analysis of fluorescent markers of basolateral endocytosis and recycling suggest that endocytosis is normal in cav-2 mutants and thus, that the suppression of basolateral recycling defects in cav-2 mutants is due to changes in intracellular trafficking pathways. Finally, cav-2 mutants also have abnormal trafficking of yolk proteins. Taken together, these data indicate that caveolin-2 is an integral component of the trafficking network in the intestinal cells of C. elegans.

Synthesis of a new conjugated 2,2′-(1,4-phenylenedivinylene)bis-quinoxaline and a series of styryl derivatives of quinoxaline and quinoline as promising electro- and photoluminescent materials

A new poly(p-phenylenedivinylene) oligomer 2,2'-(1,4-phenylenedivinylene)bisquinoxaline, a promising candidate in organic electroluminescent devices, was synthesised via a Knoevenagel condensation reaction of 2-methylquinoxaline with 1,4-benzenedicarbaldehyde. The same synthetic strategy was applied to obtain substituted styryl dyes, derivatives of quinoxaline, whereas styryl derivatives of quinoline were prepared by "Anil Synthesis". The considered dyes are of interest as fluorescence emitting materials.

ABCB19/PGP19 stabilises PIN1 in membrane microdomains in Arabidopsis

Auxin transport is mediated at the cellular level by three independent mechanisms that are characterised by the PIN-formed (PIN), P-glycoprotein (ABCB/PGP) and AUX/LAX transport proteins. The PIN and ABCB transport proteins, best represented by PIN1 and ABCB19 (PGP19), have been shown to coordinately regulate auxin efflux. When PIN1 and ABCB19 coincide on the plasma membrane, their interaction enhances the rate and specificity of auxin efflux and the dynamic cycling of PIN1 is reduced. However, ABCB19 function is not regulated by the dynamic cellular trafficking mechanisms that regulate PIN1 in apical tissues, as localisation of ABCB19 on the plasma membrane was not inhibited by short-term treatments with latrunculin B, oryzalin, brefeldin A (BFA) or wortmannin--all of which have been shown to alter PIN1 and/or PIN2 plasma membrane localisation. When taken up by endocytosis, the styryl dye FM4-64 labels diffuse rather than punctuate intracellular bodies in abcb19 (pgp19), and some aggregations of PIN1 induced by short-term BFA treatment did not disperse after BFA washout in abcb19. Although the subcellular localisations of ABCB19 and PIN1 in the reciprocal mutant backgrounds were like those in wild type, PIN1 plasma membrane localisation in abcb19 roots was more easily perturbed by the detergent Triton X-100, but not other non-ionic detergents. ABCB19 is stably associated with sterol/sphingolipid-enriched membrane fractions containing BIG/TIR3 and partitions into Triton X-100 detergent-resistant membrane (DRM) fractions. In the wild type, PIN1 was also present in DRMs, but was less abundant in abcb19 DRMs. These observations suggested a rationale for the observed lack of auxin transport activity when PIN1 is expressed in a non-plant heterologous system. PIN1 was therefore expressed in Schizosaccharomyces pombe, which has plant-like sterol-enriched microdomains, and catalysed auxin transport in these cells. These data suggest that ABCB19 stabilises PIN1 localisation at the plasma membrane in discrete cellular subdomains where PIN1 and ABCB19 expression overlaps.

Organic Styryl Dye Nanoparticles: Synthesis and Unique Spectroscopic Properties

We report the synthesis and unique spectroscopic properties of organic styryl dye nanoparticles. Aqueous-phase ion association between a cationic styryl dye 2-(4-(dimethylamino)styryl)-1-ethylpyridinium (DASPE), possessing both electron donor and acceptor groups in its molecule, and tetraphenylborate (TPB) or tetrakis(4-fluorophenyl)borate (TFPB) anion, in the presence of poly(vinylpyrrolidone), produces the ion-based dye (DASPE) nanoparticles of approximately 30-100 nm in diameter. Absorption spectra of the DASPE nanoparticles show a large bathochromic shift in comparison with that of the dye monomer in water. Quantum chemical calculations demonstrate that ion-pair formation brings about a large internal rotation around a single bond in DASPE, and this internal twisting as well as local polarity of the counteranion have a strong influence on the red shift of the optical spectra. Furthermore, nanoparticle formation results in enhanced fluorescence of DASPE: more than a 20-fold enhancement in the fluorescence quantum yield as compared to that of the dye monomer in water, giving a new methodology for the synthesis of fluorescent organic nanoparticles. The observed fluorescence would come from an intramolecular charge-transfer (ICT) excited state stabilized by the matrix of TPB or TFPB, and the enhancement is due to both the high rotational resistance for the single bond in DASPE and the matrix polarity effect that can suppress the nonradiative processes.

Synapsin IIa Controls the Reserve Pool of Glutamatergic Synaptic Vesicles

Synapsins regulate synaptic transmission by controlling the reserve pool of synaptic vesicles. Each of the three mammalian synapsin genes is subject to alternative splicing, yielding several isoforms whose roles are unknown. To investigate the function of these isoforms, we examined the synaptic effects of introducing each isoform into glutamatergic cultured hippocampal neurons from synapsin triple knock-out mice. Remarkably, we found that synapsin IIa was the only isoform that could rescue the synaptic depression phenotype of the triple knock-out mice; other isoforms examined, including the well-studied synapsin Ia isoform, had no significant effect on the kinetics of synaptic depression. The slowing of synaptic depression by synapsin IIa was quantitatively paralleled by an increase in the density of reserve pool synaptic vesicles, as measured either by fluorescent tagging of the vesicle protein synaptobrevin-2 or by staining with the styryl dye FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-diethylamino)phenyl)-hexatrienyl)pyridinium dibromide]. Our results provide further support for the hypothesis that synapsins define the kinetics of synaptic depression at glutamatergic synapses by controlling the size of the vesicular reserve pool and identify synapsin IIa as the isoform primarily responsible for this task.

Activity-dependent coordination of presynaptic release probability and postsynaptic GluR2 abundance at single synapses

The strength of an excitatory synapse depends on both the presynaptic release probability (p(r)) and the abundance of functional postsynaptic AMPA receptors. How these parameters are related or balanced at a single synapse remains unknown. By taking advantage of live fluorescence imaging in cultured hippocampal neurons where individual synapses are readily resolved, we estimate p(r) by labeling presynaptic vesicles with a styryl dye, FM1-43, while concurrently measuring postsynaptic AMPA receptor abundance at the same synapse by immunolabeling surface GluR2. We find no appreciable correlation between p(r) and the level of surface synaptic GluR2 under basal condition, and blocking basal neural activity has no effect on the observed lack of correlation. However, elevating network activity drives their correlation, which accompanies a decrease in mean GluR2 level. These findings provide the direct evidence that the coordination of pre- and postsynaptic parameters of synaptic strength is not intrinsically fixed but that the balance is tuned by synaptic use at individual synapses.

Synthesis and condensation reactions of the boron difluoride complex with 3-acetyl-4-hydroxy-1-methyl-2-quinolone

A reaction of 3-acetyl-4-hydroxy-1-methyl-2-quinolone with boron trifluoride etherate gave its boron difluoride complex. Condensation of this complex with various carbonyl compounds afforded new 2-quinolone derivatives (polymethine and styryl dyes) that intensely absorb in the visible region of the spectrum and show effective fluorescence.

Design of styryl dye single crystals in the presence of low-molecular aromatic compounds and peculiarities of [2+2]-photocycloaddition in these single crystals

A variety of crystal packing motifs for styryl dye Et-Py+-CH=CH-C6H4OMe ClO4− crystallized from solvent mixtures containing different low-molecular aromatic compounds was investigated by X-ray diffraction method. The presence of some of these aromatic compounds was found to affect the mode of the aggregation of the dye structural units in the course of crystal nucleation, regardless of whether these compounds are involved in the growing crystal. The aromatic molecules containing two proton-donating groups can be involved in the crystal packing of the dye as a solvate that forms weak hydrogen bonds with perchlorate anions or other solvate molecules. The aromatic molecules without functional groups (except for benzene) are not involved in the growing crystal as additional building blocks. The syn-head-to-tail stacking with contiguous and antiparallel arrangement of the ethylene bonds is the main packing motif of dye cations in crystals of unsolvated and solvated forms. This is favorable for accomplishing the [2+2]-photocycloaddition (PCA) reaction resulting in the centrosymmetric rctt-isomer of a cyclobutane derivative. In the solvated forms of the crystals, the solvate molecules and perchlorate anions form loose layers between the cation stacks. These layers are capable of leveling internal stresses caused by the PCA reaction, thus promoting this reaction as a single-crystal-to-single-crystal transformation. Other conditions for the PCA reaction to proceed without single crystal degradation are (1) retention of the initial crystal symmetry in the course of the PCA and (2) stack separation into dimeric pairs with d1 < 4.2 A < d2, where d1 and d2 are the distances between the ethylene bonds within the dimeric pairs and between them, respectively. The regularities found for the crystal packing and the PCA reaction in styryl dye single crystals may be used to design information recording and storage cells.

Effect of Clotrimazole on the Pump Cycle of the Na,K-ATPase

The effect of the antimycotic drug clotrimazole (CLT) on the Na,K-ATPase was investigated using fluorescence and electrical measurements. The results obtained by steady-state fluorescence experiments with the electrochromic styryl dye RH421 were combined with those achieved by a pre-steady-state method based on fast solution exchange on a solid supported membrane that adsorbs the protein. Both techniques are suitable for monitoring the electrogenic steps of the pump cycle and are in general complementary, yielding distinct kinetic information. The experiments show clearly that CLT affects specific partial reactions of the pump cycle of the Na,K-ATPase with an affinity in the low micromolar range and in a reversible manner. All results can be consistently explained by proposing the CLT-promoted formation of an ion-occluded-CLT-bound conformational E 2 state, E 2 CLT ( X 2 ) that acts as a “dead-end” side track of the pump cycle, where X stands for H + or K +. Na + binding, enzyme phosphorylation, and Na + transport were not affected by CLT, and at high CLT concentrations ∼ 1 3 of the enzyme remained active in the physiological transport mode. The presence of Na + and K + destabilized the inactivated form of the Na,K-ATPase.

FRET analysis of transmembrane flipping of FM4–64 in plant cells: is FM4–64 a robust marker for endocytosis?

Although the styryl dye FM4-64 is now used routinely to monitor endocytosis in plants, the argument about its potential to cytoplasmically and non-endocytically relocate into a selective set of vesicular compartments persists. To address this question, we determined whether fluorescence resonance energy transfer (FRET) could occur between a cytoplasmically expressed, short-wavelength excitation green fluorescent protein (GFP) and FM4-64 in Nicotiana benthaminana. After exposure to FM4-64, the root hair plasma membrane and internal organelles became labelled. Under these conditions, no FRET with cytoplasmic GFP was seen. However, if the cells were treated with a low concentration of quillajasaponin, a membrane permeabilization agent, the cells continued to stream and FRET was detected. Thereby, we demonstrate that under conditions that do not severely compromise cell viability, the FM4-64 dye becomes a suitable FRET partner for the cytoplasmically localized GFP. Under normal conditions, FM4-64 does not significantly enter the cytosolic side of the membrane, but remains at the plasma membrane or trapped in the organelles of the endocytic pathway. Hence, when the structure or permeability of the plasma membrane is unaltered, FM4-64 dye is a robust marker for endocytosis.

Lysosomes Are the Major Vesicular Compartment Undergoing Ca2+-Regulated Exocytosis from Cortical Astrocytes

Although Ca(2+)-dependent exocytosis is considered to be a pathway for gliotransmitter release from astrocytes, the structural and functional bases of this process remain controversial. We studied the relationship between near-membrane Ca(2+) elevations and the dynamics of single astroglial vesicles with styryl (FM) dyes. We show that cultured astrocytes, unlike neurons, spontaneously internalize FM dyes, resulting in the labeling of the entire acidic vesicle population within minutes. Interestingly, metabotropic glutamate receptor activation did not affect the FM labeling. Most FM-stained vesicles expressed sialin, CD63/LAMP3, and VAMP7, three markers for lysosomes and late endosomes. A subset of lysosomes underwent asynchronous exocytosis that required both Ca(2+) mobilization from intracellular stores and Ca(2+) influx across the plasma membrane. Lysosomal fusion occurred within seconds and was complete with no evidence for kiss and run. Our experiments suggest that astroglial Ca(2+)-regulated exocytosis is carried by lysosomes and operates on a timescale orders of magnitude slower than synaptic transmission.

Optical Biomedical Diagnostics: Sensors with Optical Response Based on Two-Photon Excited Luminescent Dyes for Biomolecules Detection

The spectral properties of novel styryl dyes developed for the biomacromolecules (such as DNA) detection and imaging were investigated. The energy structures of dye molecules were examined. The spectral data prove that dyes aggregate and interact with DNA. The essential increase of the fluorescence intensity of dyes in the presence of DNA was observed. The photostability and phototoxic influence on the DNA of several styryl dyes were studied by analyzing absorption, fluorescence, and phosphorescence spectra of these dyes and dye-DNA systems. Changes of the optical density value of dye-DNA solutions caused by the irradiation were fixed in the DNA and dye absorption wavelength regions. Fluorescence emission of dye-DNA complexes upon two-photon excitation at wavelength 1064 nm with the 20-nanosecond pulsed YAG:Nd3+ laser and at 840 nm with the 90 famtosecond pulsed Ti:sapphire laser was registered. The values of two-photon absorption cross-sections of dye-DNA complexes were evaluated.

Properties of the SR Ca-ATPase in an Open Microsomal Membrane Preparation

SR vesicles isolated from rabbit muscle were treated by a SDS incubation and subsequent dialysis to obtain open membrane fragments that allow a direct access to the luminal membrane surface and especially to the ion-binding sites in the P-E2 conformation of the Ca-ATPase. The open membrane fragments showed about 80% of the enzyme activity in the untreated membranes. Pump function was investigated by using electrochromic styryl dyes. The kinetic properties of cytoplasmic ion binding showed no significant differences between the Ca-ATPases in SR vesicles and in membrane fragments. From pH-dependent Ca2+ binding it could be deduced that due to the SDS treatment the density of negatively charged lipid was increased by one elementary charge per 12 lipid molecules. Major differences between Ca-ATPase from SR vesicles and membrane fragments were the respective fluorescence amplitudes. This effect is, however, produced by dye-lipid interaction and not by pump function. It was demonstrated that time-resolved kinetics may be study by the use of caged compounds such as caged ATP or caged calcium also in the case of the membrane fragments.

SDF 1-alpha (CXCL12) triggers glutamate exocytosis from astrocytes on a millisecond time scale: Imaging analysis at the single-vesicle level with TIRF microscopy

Chemokines are small chemotactic molecules widely expressed throughout the central nervous system. A number of papers, during the past few years, have suggested that they have physiological functions in addition to their roles in neuroinflammatory diseases. In this context, the best evidence concerns the CXC-chemokine stromal cell-derived factor (SDF-1α or CXCL12) and its receptor CXCR4, whose signalling cascade is also implicated in the glutamate release process from astrocytes. Recently, astrocytic synaptic like microvesicles (SLMVs) that express vesicular glutamate transporters (VGLUTs) and are able to release glutamate by Ca 2+-dependent regulated exocytosis, have been described both in tissue and in cultured astrocytes. Here, in order to elucidate whether SDF-1α/CXCR4 system can participate to the brain fast communication systems, we investigated whether the activation of CXCR4 receptor triggers glutamate exocytosis in astrocytes. By using total internal reflection (TIRF) microscopy and the membrane-fluorescent styryl dye FM4-64, we adapted an imaging methodology recently developed to measure exocytosis and recycling in synaptic terminals, and monitored the CXCR4-mediated exocytosis of SLMVs in astrocytes. We analyzed the co-localization of VGLUT with the FM dye at single-vesicle level, and observed the kinetics of the FM dye release during single fusion events. We found that the activation of CXCR4 receptors triggered a burst of exocytosis on a millisecond time scale that involved the release of Ca 2+ from internal stores. These results support the idea that astrocytes can respond to external stimuli and communicate with the neighboring cells via fast release of glutamate.

Synthesis, spectral characterisation and anti‐bacterial activity of various 1,3,4‐oxadiazine cyanine dyes

A number of new styryl, aza-styryl, acyclic mero, cyclic mero and mixed cyanine dyes with a benzo[2,3-e; 2′,3′-e′]bis-1,3,4-oxadiazine nucleus were synthesised. The absorption spectra in the visible region of all the synthesised cyanine dyes in 95% ethanol solution were examined and studied in detail. Anti-bacterial activity for a number of selected dyes was evaluated against various bacterial strains. The structural determination was carried out using elemental and spectroscopic analysis.

Magnitude and direction of vesicle dynamics in growing pollen tubes using spatiotemporal image correlation spectroscopy and fluorescence recovery after photobleaching.

The delivery of cell wall material and membrane to growing plant cell surfaces requires the spatial and temporal coordination of secretory vesicle trafficking. Given the small size of vesicles, their dynamics is difficult to quantify. To quantitatively analyze vesicle dynamics in growing pollen tubes labeled with the styryl dye FM1-43, we applied spatiotemporal correlation spectroscopy on time-lapse series obtained with high-speed confocal laser scanning microscopy recordings. The resulting vector maps revealed that vesicles migrate toward the apex in the cell cortex and that they accumulate in an annulus-shaped region adjacent to the extreme tip and then turn back to flow rearward in the center of the tube. Fluorescence recovery after photobleaching confirmed vesicle accumulation in the shoulder of the apex, and it revealed that the extreme apex never recovers full fluorescence intensity. This is consistent with endocytotic activity occurring in this region. Fluorescence recovery after photobleaching analysis also allowed us to measure the turnover rate of the apical vesicle population, which was significantly more rapid than the theoretical rate computed based on requirements for new cell wall material. This may indicate that a significant portion of the vesicles delivered to the apex does not succeed in contacting the plasma membrane for delivery of their contents. Therefore, we propose that more than one passage into the apex may be needed for many vesicles before they fuse to the plasma membrane and deliver their contents.