Dicyanovinyl)julolidine Research Articles

Use of a fluorescent molecular rotor probe for nanoplastics assessment in epiphytic biofilms growing on submerged vegetation of Lake Saint Pierre (St. Lawrence River)

Studies on plastic pollution conducted in freshwaters mainly focused on monitoring plastic debris in the water column or in the sediments. Few studies have investigated the occurrence of plastic debris in benthic biofilms (periphyton). Yet, algal biofilms may potentially act as a sink for plastic debris, trapping it within their mucilaginous or filamentous matrix. Biofilms may also represent a source of plastic debris by sloughing when they become senescent. In addition, plastic debris accumulated within biofilms may enter the food web via primary consumers. Considering these observations, this study aims to quantify nanoplastics (NPs) accumulated in biofilms growing on aquatic vegetation from Lake Saint Pierre (LSP), a fluvial lake of the St. Lawrence River, and its archipelago. Biofilms were removed from submerged plants and the presence of NPs was assessed by spectrophotometry using the fluorescent molecular rotor probe 9(dicyanovinyl)-julolidine (DCVJ). The results of this study confirm the biofilms’ ability to act as a sink for NPs. Despite the fact that the determination of the absolute nanoparticle number and size distribution remains a challenge, we estimated a median concentration of 1.05 × 109 NP/mg of biofilm dry weight (DW) when using 100 nm polystyrene beads for calibration. Concentrations were significantly different between water masses, with higher concentrations in samples collected in the two lateral water masses compared to the central water mass. Our study provides, for the first time, a quantitative assessment of NPs from epiphytic biofilms in a large river under the influence of anthropogenic sources.

Path-dependent rheology of carbon particle-hydroxyethylcellulose fluids

Polymeric fluids with tunable viscosity find application in different sectors, ranging from enhanced oil recovery to environmental remediation. This study investigates polymeric fluids obtained with hydroxyethylcellulose (HEC) and biocarbon particles prepared at low (650⁰C, LM) and high (900⁰C, HM) temperature of pyrolization. High pyrolization temperatures increased the hydrophobicity of biocarbon particles. In the absence of HEC, HM particles partitioned preferentially in non-polar solvents (e.g. toluene, rather than in water). Without particles, the shear loss (G”) and storage (G’) moduli of aqueous hydroxyethylcellulose solutions slowly increased over time, due to HEC hydration. The progressive increase in molecular crowding and microviscosity due to the hydration of HEC was demonstrated using steady-state fluorescence spectroscopy with the molecular rotor 9-(dicyanovinyl)julolidine (DCVJ) as a probe. Particles added to HEC increased the viscoelastic moduli of HEC solutions only slightly (i.e. less than doubled them) when HEC was pre-hydrated, and significantly when HEC was not pre-hydrated. Specifically, addition of LM and HM particles (1−30 g/L) to non-hydrated HEC (16 g/L) increased the viscoelastic moduli of HEC solutions in water from below detection to G”>9 Pa and G’>6 Pa (in the whole range of biocarbon particle concentrations analyzed). Our study investigates for the first time the effect of path-dependence of the preparation method on the rheology of HEC-carbon particle fluids. The viscoelastic moduli increase of HEC is ascribed to hydrophobic interactions between biocarbon particles and non-hydrated HEC. The preferential particle partitioning in aqueous HEC solutions (rather than in toluene) is also attributed to these interactions. Such interactions were attributed to the partially hydrophobic nature of HEC, which partitioned at the air-water interface (rather than exclusively in bulk water), lowering the interfacial tension and producing interfacial films with compressional rigidity. HEC interfacial film rigidity changed upon particle addition, supporting the hypothesis of HEC-particle interactions. Compression isotherms modelled using the theory by Marczak et al. were used here for the first time as an indirect tool to gain insights regarding HEC-carbon particle interactions. Partial HEC hydrophobicity was also indicated by the higher viscosity measured in ethanol-water solutions than in water, which showed that ethanol-water was a better solvent than pure water. Steady state fluorescence measurements confirmed that the amount of HEC-bound solvent was greater in ethanol-water mixtures than in pure water. Biocarbon particles mixed with non-hydrated HEC were well-dispersed. Instead, biocarbon particles aggregated when they were mixed with hydrated HEC, indicating that the magnitude of attractive forces between particles and HEC decreased when HEC was hydrated. Compression isotherms measured at the air-water interface with non-hydrated HEC alone and with non-hydrated HEC-biocarbon particle mixtures differed, indicating HEC-particle co-adsorption at the air-water interface and the formation of HEC-particle aggregates.

Detection of polystyrene nanoplastics in biological tissues with a fluorescent molecular rotor probe.

The release of nanoplastics (NPs) from the weathering and degradation of plastics is an important environmental concern. Given their small sizes and their invasiveness in cells, methods for the detection of NPs in biological tissues are urgently needed. A simple fluorescence-based methodology for the detection of polystyrene NPs in biological tissues is proposed. The commercially available molecular rotor probe 9- (dicyanovinyl)-julolidine (DCVJ) has the properties to detect changes in hydrophobicity and microviscosity and was used to detect NPs. Increasing concentrations of 50 and 100 nm NPs in water and in tissue extracts were mixed with the DCVJ probe and the emission spectra determined between 480-800 nm at 450 nm excitation. The data revealed that NPs induces a second emission peak at 620 nm that differed from the normal spectra of the biological extract at 500 nm. A significant linear relationship was obtained for NPs of both sizes (r=0.98; P<0.001) with a theoretical limit of detection of 65 ng/mL. A simple and rapid microplate spectrofluorometric method for the semi-quantitative detection of polystyrene NPs in biological tissues is thus presented.

Mechanistic Insight into the Binding Profile of DCVJ and α-Synuclein Fibril Revealed by Multiscale Simulations.

Parkinson's disease (PD) is a serious neurodegenerative disease and is characterized by abnormal α-synuclein (α-syn) accumulation in Lewy bodies (LB) and Lewy neurites (LN), which makes α-syn an important imaging target for PD. An imaging probe that quantifies fibrillar α-syn can enhance the clinical diagnosis of PD and can also be used to evaluate the efficacy of therapeutics aimed at reducing the abnormal aggregation of the α-syn fibril in the brain. In this paper, we study the binding profile of fibrillar α-syn with a fluorescent probe 4-(dicyanovinyl)julolidine (DCVJ), which is being explored for identifying α-syn imaging agents. A multiscale simulation workflow including molecular docking, molecular dynamics, metadynamics, and QM/MM calculations was implemented. We find that DCVJ can bind to multiple sites of α-syn which are located either at the surface or in the core. Free energy calculations using implicit solvent models reveal that the most favorable binding mode for DCVJ is associated with the core binding site and is further confirmed by metadyamics simulation. Besides, a dynamic binding pathway is discovered, which reveals that DCVJ binds gradually into the core of the fibril passing through several intermediate states. The conformational arrest of the dicyano vinyl group in the fibrillar environment could explain the reason behind the fibril-specific fluorescence of DCVJ. Furthermore, based on hybrid QM/MM calculations, the molecular geometry of the dicyano vinyl group is found to be environment specific which explains why DCVJ serves as a staining agent for such fibrillar-like environments. Our results could be helpful for elucidating the binding mechanism of imaging tracers with the fibrillar form of α-syn and explain their fibrillar-specific optical properties, a knowledge that in turn can be used to guide the design and development of compounds with higher affinity and selectivity for α-syn using structure-based strategies.

Fluorescent Probe DCVJ Shows High Sensitivity for Characterization of Amyloid β‐Peptide Early in the Lag Phase

The aggregation of intrinsically disordered and misfolded proteins in the form of oligomers and fibrils plays a crucial role in a number of neurological and neurodegenerative diseases. Currently, most probes and biophysical techniques that detect and characterize fibrils at high resolution fail to show sensitivity and binding for oligomers. Here, we show that 9-(dicyano-vinyl)julolidine (DCVJ), a class of molecular rotor, binds amyloid beta (Aβ) early aggregates, and we report the kinetics as well as packing of the oligomer formation. The binding of DCVJ to Aβ40 increased its emission intensity with time at 510 nm and produced a second excimer peak at 575 nm. However, DCVJ did not bind to the prefibrillar aggregates of Aβ42, which indicated that the oligomers formed by Aβ40 and Aβ42 were not the same. The F4C F19W mutant of Aβ40, which did not form fibrils, also bound DCVJ, but the emission spectral profile varied from that of the wild-type (WT). Atomic force microscopy images of WT Aβ40, the F4C F19W mutant, and Aβ42 oligomers displayed differences in size and shape, confirming the difference in their DCVJ spectra. The effect of epigallocatechin-3-gallate (EGCG) on the reduction of Aβ42 fibrils was also observed with finer detail than with other techniques. The results of this study show that DCVJ detects early aggregates and provides valuable information regarding the oligomer kinetics, packing, and mechanism of formation.

High stability and cooperative unfolding of α-synuclein oligomers.

Many neurodegenerative diseases are linked with formation of amyloid aggregates. It is increasingly accepted that not the fibrils but rather oligomeric species are responsible for degeneration of neuronal cells. Strong evidence suggests that in Parkinson's disease (PD), cytotoxic α-synuclein (αSN) oligomers are key to pathogenicity. Nevertheless, insight into the oligomers' molecular properties remains scarce. Here we show that αSN oligomers, despite a large amount of disordered structure, are remarkably stable against extreme pH, temperature, and even molar amounts of chemical denaturants, though they undergo cooperative unfolding at higher denaturant concentrations. Mutants found in familial PD lead to slightly larger oligomers whose stabilities are very similar to that of wild-type αSN. Isolated oligomers do not revert to monomers but predominantly form larger aggregates consisting of stacked oligomers, suggesting that they are off-pathway relative to the process of fibril formation. We also demonstrate that 4-(dicyanovinyl)julolidine (DCVJ) can be used as a specific probe for detection of αSN oligomers. The high stability of the αSN oligomer indicates that therapeutic strategies should aim to prevent the formation of or passivate rather than dissociate this cytotoxic species.

High-Throughput Melting-Temperature Analysis of a Monoclonal Antibody by Differential Scanning Fluorimetry in the Presence of Surfactants

Differential scanning fluorimetry (DSF) is successfully used as a high-throughput screening method for the analysis of the protein melting temperature (T(m)) in the development of therapeutic monoclonal antibody (MAb) formulations. Typically, surfactants are utilized in MAb formulations as a stabilizer, but the commonly applied polarity-sensitive dye SYPRO® Orange shows bright fluorescence in the presence of micelles, concealing the signal of protein unfolding. Studying various MAb formulations containing polysorbate 20, polysorbate 80, or poloxamer 188 (PX 188), the molecular rotor probe 4-(dicyanovinyl)julolidine (DCVJ) was investigated. Although limited to higher MAb concentrations, DCVJ enabled the determination of T(m) in many formulations where SYPRO® Orange failed. It is important to note that careful background correction of placebo formulations is essential for the precise determination of T(m) and especially T(m onset). Thermal shifts of T(m1) (lowest observed thermal transition) indicating stabilizing or destabilizing effects of pH or excipient were in good agreement across all tested formulations and correlated well with differential scanning calorimetry measurements. Additionally, the micellization temperature of PX 188 was confirmed, which leads to a nonproteinous transition. With this new method, it is possible to apply DSF during the development of therapeutic proteins in surfactant-containing formulations.

Modulation of excimer formation of 9-(dicyano-vinyl)julolidine by the macrocyclic hosts

This article reports the alteration of the excited state photophysics of a molecular rotor, namely 9-(dicyano-vinyl)julolidine (DCVJ), which has been extensively used to report protein aggregation and protein conformational changes, by the various cavity sizes of cyclodextrin (CD) macrocyclic hosts, with the help of steady state, time-resolved fluorescence techniques. It is observed that, in the presence of α-CD, the characteristic features of both the monomer and excimer emissions of DCVJ are almost unperturbed. However, in the presence of β-CD, the excited photophysics of the molecule is significantly perturbed, and it is noted that β-CD inhibits the excimer formation drift of DCVJ by incorporation of a DCVJ monomer inside its cavity. The most striking findings are observed in the case of γ-CD. Initially, the excimer peak intensity drops and the monomer intensity increases, due to the 1 : 1 DCVJ/γ-CD inclusion complex formation. Above a certain concentration, another DCVJ molecule is accommodated inside the γ-CD cavity and forms an excimer, which is reflected in the intensification of the excimer peak. At higher γ-CD concentration the fluorescence intensity of the excimer shoots up, due to the formation of 2 : 2 host-guest complex, in which an additional γ-CD molecule provides extra stabilization to the excimer. Insight on the molecular picture of this host-guest interaction has been provided by docking studies followed by quantum chemical calculations.

CCVJ Is Not a Simple Rotor Probe

The photochemistry of the rotor probe 9-(2-carboxy-2-cyanovinyl)julolidine (CCVJ) was studied to elucidate a curious effect of fluid flow previously reported. The apparent sensitivity to fluid motion observed in CCVJ but not in the closely related molecule 9-(dicyanovinyl)julolidine (DCVJ) is found to be an indirect effect of a photoisomerization reaction. The results presented here demonstrate that it is this isomerization, rather than the commonly assumed TICT process, that confers viscosity-sensing ability on these fluorophores. In micromolar solutions in hydroxylic solvents CCVJ exists primarily in the carboxylate form. Only the E isomer of this anion is initially present in solutions prepared from the solid, but in room light such solutions rapidly achieve a photostationary state in which the E isomer and an essentially nonfluorescent Z isomer exist in comparable concentrations. The Z isomer is metastable in S(0) such that in the absence of light the solution reverts slowly to pure E. Unlike DCVJ where only a single isomer is possible, the production of long-lived photoproducts in CCVJ and other asymmetrically substituted styryenyl probes complicates their fluorescence response. Considerable care is needed when such fluorphores are used as steady-state sensors of environmental fluidity are used.

Thiophene‐Inserted Aryl–Dicyanovinyl Compounds: The Second Generation of Fluorescent Molecular Rotors with Significantly Redshifted Emission and Large Stokes Shift

AbstractFluorescent molecular rotors can be used as molecular sensors for the viscosity of a microenvironment. However, these molecular rotors are limited to 9‐(dicyanovinyl)julolidine (DCVJ) and a few derivatives. Furthermore, these traditional rotors show short absorption/emission wavelengths and small Stokes shifts. To address these drawbacks, we have developed a small library of new molecular rotors for viscosity sensing, prepared by incorporating a thiophene unit into the conventional fluorescent molecular rotors with the aim of accessing molecular rotors with redshifted excitation/emission wavelengths and larger Stokes shifts compared with the known rotors. The new rotors show substantially improved photophysical properties. For example, rotor 4 shows absorption/emission wavelengths of 559/697 nm, respectively, and a very large Stokes shift of 138 nm compared with the absorption/emission wavelengths (465/503 nm) and very small Stokes shift (38 nm) of the traditional fluorescent molecular rotor DCVJ. The photophysical properties of the rotors were rationalized by DFT calculations.

Free Volume Dependence of the Internal Rotation of a Molecular Rotor Probe in Room Temperature Ionic Liquids

The fluorescence efficiency of a well-known microviscosity probe, 9-(dicyanovinyl)julolidine (DCVJ), which is highly sensitive to the viscosity of the medium, has been studied in seven imidazolium ionic liquids (ILs) of varying viscosities over a temperature range of 10-60 degrees C. The microviscosities around the probe in different ILs have been estimated from the linear dependence of the logarithm of fluorescence quantum yield (log phi(f)) on the logarithm of the bulk viscosity (log eta) in various conventional solvents of different viscosities at room temperature. These microviscosities, which represent the local environments around the probe, are found to be significantly different from the directly measured bulk viscosities of these ILs. The log phi(f) vs log (eta/T) plots, which are also expected to be linear, interestingly show a bilinear behavior in more viscous ILs with a break around 28-30 degrees C. The observation of a similar break in the Arrhenius plots of the rate constant of the internal rotation in DCVJ and absence of any such break in the temperature dependence of the mobility of the ILs allow us to determine the important role of the free volume around the probe in dictating the nonradiative deactivation rate or the fluorescence efficiency of DCVJ. The break in the plots, which implies a change in the available free volume around the probe at approximately 28-30 degrees C, presumably arises from the repositioning of the probe from one environment to a different one of these microheterogeneous ILs with change of temperature.

A Molecular Rotor Based on an Unhindered Boron Dipyrromethene (Bodipy) Dye

This work describes a fluorescent probe for following changes in the viscosity of the surrounding medium. The optical properties, fluorescence characteristics, and sensitivity to frictional forces with the surrounding medium are superior to the most commonly used molecular probe, namely dicyanovinyl julolidine. The photophysical properties of the target molecule have been recorded in a range of solvents under ambient conditions, over a wide temperature range, and as a function of applied pressure. The mechanism by which the probe responds to changes in local viscosity involves gyration of the meso-phenylene ring and accompanying distortion of the dipyrrin framework, as indicated by molecular dynamics simulations. Indeed, temperature-dependence measurements have established that the activation energy is small when the solvent viscosity is relatively low, but there is a turnover to strong activation control at very high viscosity. A small but definite solvent dependence appears when the viscosity is varied ...

Detection and Characterization of Aggregates, Prefibrillar Amyloidogenic Oligomers, and Protofibrils Using Fluorescence Spectroscopy

Transthyretin (TTR) is a protein linked to a number of different amyloid diseases including senile systemic amyloidosis and familial amyloidotic polyneuropathy. The transient nature of oligomeric intermediates of misfolded TTR that later mature into fibrillar aggregates makes them hard to study, and methods to study these species are sparse. In this work we explore a novel pathway for generation of prefibrillar aggregates of TTR, which provides important insight into TTR misfolding. Prefibrillar amyloidogenic oligomers and protofibrils of misfolded TTR were generated in vitro through induction of the molten globule type A-state from acid unfolded TTR through the addition of NaCl. The aggregation process produced fairly monodisperse oligomers (300-500 kD) within 2 h that matured after 20 h into larger spherical clusters (30-50 nm in diameter) and protofibrils as shown by transmission electron microscopy. Further maturation of the aggregates showed shrinkage of the spheres as the fibrils grew in length, suggesting a conformational change of the spheres into more rigid structures. The structural and physicochemical characteristics of the aggregates were investigated using fluorescence, circular dichroism, chemical cross-linking, and transmission electron microscopy. The fluorescent dyes 1-anilinonaphthalene-8-sulfonate (ANS), 4-4-bis-1-phenylamino-8-naphthalene sulfonate (Bis-ANS), 4-(dicyanovinyl)-julolidine (DCVJ), and thioflavin T (ThT) were employed in both static and kinetic assays to characterize these oligomeric and protofibrillar states using both steady-state and time-resolved fluorescence techniques. DCVJ, a molecular rotor, was employed for the first time for studies of an amyloidogenic process and is shown useful for detection of the early steps of the oligomerization process. DCVJ bound to the early prefibrillar oligomers (300-500 kD) with an apparent dissociation constant of 1.6 muM, which was slightly better than for ThT (6.8 muM). Time-resolved fluorescence anisotropy decay of ANS was shown to be a useful tool for giving further structural and kinetic information of the oligomeric aggregates. ThT dramatically increases its fluorescence quantum yield when bound to amyloid fibrils; however, the mechanism behind this property is unknown. Data from this work suggest that unbound ThT is also intrinsically quenched and functions similarly to a molecular rotor, which in combination with its environmental dependence provides a blue shift to the characteristic 482 nm wavelength when bound to amyloid fibrils.

The Susceptibility of Non-UV Fluorescent Membrane Dyes to Dynamical Properties of Lipid Membranes

Fluorescence spectroscopy and microscopy are powerful techniques to detect dynamic properties in artificial and natural lipid membrane systems. Unfortunately, most fluorescent dyes that sense dynamically relevant membrane parameters are UV sensitive. Their major disadvantage is a high susceptibility to fluorescence bleaching. Additionally, the risk for hazardous damages in biological components generally increases with decreasing excitation wavelength. Therefore the use of non-UV–sensitive membrane dyes would provide significant advantage, particularly for applications in fluorescence microscopy, which usually implies high local excitation intensities. We applied steady-state fluorescence spectroscopy techniques to several UV and non-UV membrane dyes to detect and compare dynamically relevant excitation and emission characteristics. Small unilamellar liposomes (composed of egg yolk phosphatidylcholine) served as a model system for biological membranes. The dynamic properties of the membranes were varied by two independent parameters: the intrinsic cholesterol content (0–50 mol%) and temperature (10–50°C). We tested four non-UV–sensitive membrane dyes: 9-diethylamino-5H-benzophenoxazine-5-one (Nile Red), 4-(dicyanovinyl)julolidine (DCVJ), N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide (FM 4-64), and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiIC18). We also tested three derivatives of DiIC18: DiIC16 and DiIC12 differ in acyl chain length and Fast-DiIC18 provides double bonds between hydrocarbon atoms. The spectral results were compared to established fluorescence characteristics of four UV membrane dyes: the anisotropy of 1-6-phenyl-1,3,5,-hexatrien (DPH), two derivatives of DPH (TMA-DPH and COO−-DHP), and the generalized polarization of 6-dodecanoyl-2-dimethyl-aminonaphthalene (Laurdan). Our results indicate that the tested non-UV dyes do not reveal dynamically relevant membrane parameters in a direct manner. However, spectral characteristics make DiIC18, Nile Red, and DCVJ promising probes for the microscopic detection of lateral lipid organization, an indirect indicator of membrane dynamics. In particular, DiIC18 showed very selective shifts in the emission spectra at defined temperatures and cholesterol contents that have not been reported elsewhere.

New fluorescent probes for the measurement of cell membrane viscosity

Background: Molecular rotors are fluorescent molecules that exhibit viscosity-dependent fluorescence quantum yield, potentially allowing direct measurements of cell membrane viscosity in cultured cells. Commercially available rotors, however, stain not only the cell membrane, but also bind to tubulin and migrate into the cytoplasm. We synthesized molecules related to 9-(dicyanovinyl)-julolidine (DCVJ), which featured hydrocarbon chains of different length to increase membrane compatibility. Results: Longer hydrocarbon chains attached to the fluorescent rotor reduce the migration of the dye into the cytoplasm and internal compartments of the cell. The amplitude of the fluorescence response to fluid shear stress, known to decrease membrane viscosity, is significantly higher than the response obtained from DCVJ. Notably a farnesyl chain showed a more than 20-fold amplitude over DCVJ and allowed detection of membrane viscosity changes at markedly lower shear stresses. Conclusions: The modification of molecular rotors towards increased cell membrane association provides a new research tool for membrane viscosity measurements. The use of these rotors complements established methods such as fluorescence recovery after photobleaching with its limited spatial and temporal resolution and fluorescence anisotropy, which has low sensitivity and may be subject to other effects such as deformation.

Fluid shear stress increases membrane fluidity in endothelial cells: a study with DCVJ fluorescence.

Fluid shear stress (FSS) has been shown to be an ubiquitous stimulator of mammalian cell metabolism. Although many of the intracellular signal transduction pathways have been characterized, the primary mechanoreceptor for FSS remains unknown. One hypothesis is that the cytoplasmic membrane acts as the receptor for FSS, leading to increased membrane fluidity, which in turn leads to the activation of heterotrimetric G proteins (13). 9-(Dicyanovinyl)-julolidine (DCVJ) is a fluorescent probe that integrates into the cell membrane and changes its quantum yield with the viscosity of the environment. In a parallel-plate flow chamber, confluent layers of DCVJ-labeled human endothelial cells were exposed to different levels of FSS. With increased FSS, a reduced fluorescence intensity was observed, indicating an increase of membrane fluidity. Step changes of FSS caused an approximately linear drop of fluorescence within 5 s, showing fast and almost full recovery after shear cessation. A linear dose-response relationship between shear stress and membrane fluidity changes was observed. The average fluidity increase over the entire cell monolayer was 22% at 26 dyn/cm(2). This study provides evidence for a link between FSS and membrane fluidity, and suggests that the membrane is an important flow mechanosensor of the cell.

An initial signal of activation of rat peritoneal mast cells stimulated by Datura stramonium agglutinin: a confocal fluorescence microscopic analysis of intracellular calcium ion and cytoskeletal assembly.

A confocal fluorescence microscope using fluo-3 and 9-(dicyanovinyl)- julolidine (DCVJ) was used to study the mast cell activation by the N-acetyl glucosamine oligomer specific lectin Datura stramonium agglutinin (DSA) and inhibition by antagonist lectins having affinity to N-acetyl glucosamine (GlcNAc). DSA induced a transient increase in intracellular free calcium concentration ([Ca2+]i) followed by cytoskeletal disassembly and reassembly in rat peritoneal mast cells. These changes induced by DSA resulted in histamine release. The time course of fluorescence intensity in mast cells loaded with fluo-3- or DCVJ and activated by DSA resembled those activated by the basic polymer compound 48/80. Inhibition of [Ca2+]i rise by antagonist lectins was responsible for the inhibition of cytoskeletal assembly and the consequent histamine release induced by DSA. At the level of the individual cell, a mast cell stimulated by DSA responds in an all-or-none fashion. DSA possible induced intracellular calcium mobilization and cytoskeletal change by recognizing the GlcNAc-oligomer residues of specific glycoproteins of mast cells.

A fluorescent molecular rotor probes the kinetic process of degranulation of mast cells.

A confocal fluorescence microscope was used to study the exocytotic secretory processes of mast cells in combination with an fluorescent molecular rotor, 9-(dicyanovinyl)julolidine (DCVJ). DCVJ is known to be an unique fluorescent dye which increases its quantum yield with decreasing intramolecular rotation. Here, DCVJ-loaded peritoneal rat mast cells were stimulated with compound 48/80 and their fluorescence images were compared with fluorescence calcium images of fluo-3-loaded mast cells. Subsequent to transient increases in intracellular free calcium ion concentration, DCVJ fluorescence increased dramatically in the cytoplasm and formed a ring-like structure around the nucleus, suggesting the possibility that the dye bound to the proteins composing the cytoskeletal architecture. Furthermore, the increases of DCVJ fluorescence intensities were mostly blocked in the presence of cytochalasin D (10 microM). However, fluo-3 fluorescence intensities still increased after addition of compound 48/80.

9-(Dicyanovinyl)Julolidine Binding to Bovine Brain Calmodulin

A molecular rotor, 9-(dicyanovinyl)julolidine (DCVJ), is a fluorescent dye whose intramolecular rotation determines its fluorescence yield [Kung, C.E. & Reed, J.K. (1989) Biochemistry 28, 6678-6686]. DCVJ binds to bovine brain calmodulin and emits strong fluorescence. In fluorescence titration experiments, the dissociation constant and the number of binding sites were determined to be 20 +/- 10 microM and 0.7 +/- 0.5 in the presence of Ca2+, and 22 +/- 10 microM and 0.6 +/- 0.5 in the absence of Ca2+, respectively. The fluorescence intensity of bound DCVJ increased 10-fold in the presence of Ca2+ compared to that in the absence of Ca2+. The fluorescence titration curve of DCVJ-calmodulin showed a transition at pCa 6.5. Over the same Ca2+ range, a decrease in molecular ellipticity at 222 nm and an increase in tyrosine fluorescence of calmodulin were observed. These results mean that the conformational change of calmodulin due to the Ca2+ binding induces the microenvironmental change of the DCVJ binding site from the flexible to the rigid state, resulting in inhibition of the intramolecular rotation of DCVJ and an increase in its fluorescence.

Fluorescent molecular rotors: a new class of probes for tubulin structure and assembly.

9-(Dicyanovinyl)julolidine (DCVJ) is a fluorescent dye whose intramolecular rotational relaxation is solvent dependent. Since its quantum yield increases with decreasing free volume, this molecule has been very useful in monitoring synthetic polymer reactions and measuring local microviscosity changes in phospholipid bilayers [Loutfy, R. O. (1986) Pure Appl. Chem. 58, 1239-1248; Kung, C. E., & Reed, J. K. (1986) Biochemistry 25, 6114-6121]. We have used DCVJ to follow the polymerization of tubulin, a protein that can assemble into a variety of polymorphic microstructures. DCVJ binding to free tubulin is accompanied by an increase in quantum yield, indicating that DCVJ has become partially immobilized. At 4 degrees C, DCVJ binds to a single population of high-affinity hydrophobic sites (Kd = 1.12 +/- 0.26 microM) with a stoichiometry that is protein concentration dependent. n, the number of moles of DCVJ bound per mole of alpha beta dimer, approaches 1 at concentrations less than or equal to 0.5 mg/mL but decreases to a lower limit of approximately 0.3 at concentrations greater than or equal to 2.0 mg/mL. The quantum yield also increases with increasing protein concentration. This trend is unaltered by the presence of microtubule-associated proteins. These results are analyzed in terms of a concentration-dependent oligomerization of tubulin at 4 degrees C. When tubulin is polymerized at 37 degrees C to microtubules or to sheets in the presence of Zn2+, the fluorescence intensity of DCVJ increases although the magnitude of this increase differs significantly. We are able to use the distinct fluorescent and binding characteristics of the bound dye to distinguish between these two polymorphs on a molecular level.