Characterization of changes in the viscosity of lipid membranes with the molecular rotor FCVJ

Membrane viscosity is a key parameter in cell physiology, cell function, and cell signaling. The most common methods to measure changes in membrane viscosity are fluorescence recovery after photobleaching (FRAP) and fluorescence anisotropy. Recent interest in a group of viscosity sensitive fluorophores, termed molecular rotors, led to the development of the highly membrane-compatible (2-carboxy-2-cyanovinyl)-julolidine farnesyl ester (FCVJ). The purpose of this study is to examine the fluorescent behavior of FCVJ in model membranes exposed to various agents of known influence on membrane viscosity, such as alcohols, dimethyl sulfoxide (DMSO), cyclohexane, cholesterol, and nimesulide. The influence of key agents (propanol and cholesterol) was also examined using FRAP, and backcalculated viscosity change from FCVJ and FRAP was correlated. A decrease of FCVJ emission was found with alcohol treatment (with a strong dependency on the chain length and concentration), DMSO, and cyclohexane, whereas cholesterol and nimesulide led to increased FCVJ emission. With the exception of nimesulide, FCVJ intensity changes were consistent with expected changes in membrane viscosity. A comparison of viscosity changes computed from FRAP and FCVJ led to a very good correlation between the two experimental methods. Since molecular rotors, including FCVJ, allow for extremely easy experimental methods, fast response time, and high spatial resolution, this study indicates that FCVJ may be used to quantitatively determine viscosity changes in phospholipid bilayers.     

Phospholipid-bound molecular rotors: synthesis and characterization

Molecular rotors are fluorescent molecules with a viscosity-sensitive quantum yield that are often used to measure viscosity changes in cell membranes and liposomes. However, commercially available molecular rotors, such as DCVJ (1) do not localize in cell membranes but rapidly migrate into the cytoplasm leading to unreliable measurements of cell membrane viscosity. To overcome this problem, we synthesized molecular rotors covalently attached to a phospholipid scaffold. Attaching the rotor group to the hydrophobic end of phosphatidylcholine (PC) did not affect the rotor's viscosity sensitivity and allowed adequate integration into artificial bilayers as well as complete localization in the plasma membrane of an endothelial cell line. Moreover, these new rotors enabled the monitoring of phospholipid transition temperature. However, attachment of the rotor groups to the hydrophilic head of the phospholipid led to a partial loss of viscosity sensitivity. The improved sensitivity and exclusive localization in the cell plasma membrane exhibited by the phospholipid-bound molecular rotors suggest that these probes can be used for the study of membrane microviscosity.     

Detection of liposome membrane viscosity perturbations with ratiometric molecular rotors

Molecular rotors are a form of fluorescent intramolecular charge-transfer complexes that can undergo intramolecular twisting motion upon photoexcitation. Twisted-state formation leads to non-radiative relaxation that competes with fluorescence emission. In bulk solutions, these molecules exhibit a viscosity-dependent quantum yield. On the molecular scale, the fluorescence emission is a function of the local free volume, which in turn is related to the local micro-viscosity. Membrane viscosity, and the inverse; fluidity, are characteristic terms used to describe the ease of movement withing the membrane. Often, changes in membrane viscosity govern intracellular processes and are indicative of a disease state. Molecular rotors have been used to investigate viscosity changes in liposomes and cells, but accuracy is affected by local concentration gradients and sample optical properties. We have developed self-calibrating ratiometric molecular rotors to overcome this challenge and integrated the new molecules into a DLPC liposome model exposed to the membrane-fluidizing agent propanol. We show that the ratiometric emission intensity linearly decreases with the propanol exposure and that the ratiometric intensity is widely independent of the total liposome concentration. Conversely, dye concentration inside liposomes influences the sensitivity of the system. We suggest that the new self-calibrating dyes can be used for real-time viscosity sensing in liposome systems with the advantages of lifetime measurements, but with low-cost steady-state instrumentation.     

Membrane microviscosity regulates endothelial cell motility

Endothelial cell (EC) movement is an initiating and rate-limiting event in the neogenesis and repair of blood vessels. Here, we explore the hypothesis that microviscosity of the plasma membrane (PM) is a key physiological regulator of cell movement. Aortic ECs treated with membrane-active agents, such as alpha-tocopherol, cholesterol and lysophospholipids, exhibited a biphasic dependency on membrane microviscosity, in which moderate increases enhanced EC migration, but increases beyond a threshold markedly inhibited migration. Surprisingly, angiogenic growth factors, that is, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), also increased membrane microviscosity, as measured in live cells by fluorescence recovery after photobleaching (FRAP). The localization of Rac to the PM was modified in cells treated with membrane-active agents or growth factors, suggesting a molecular mechanism for how membrane microviscosity influences cell movement. Our data show that angiogenic growth factors, as well as certain lipophilic molecules, regulate cell motility through alterations in membrane properties and the consequent relocalization of critical signalling molecules to membranes.     

HaCaT keratinocytes exhibit a cholesterol and plasma membrane viscosity gradient during directed migration

Keratinocyte migration plays an important role in cutaneous wound healing by supporting the process of reepithelialisation. During directional migration cells develop a polarised shape with an asymmetric distribution of a variety of signalling molecules in their plasma membrane. Here, we investigated front-to-back differences of the physical properties of the plasma membrane of migrating keratinocyte-like HaCaT cells. Using FRAP and fluorescence lifetime analysis, both under TIR illumination, we demonstrate a reduced viscosity of the plasma membrane in the lamellipodia of migrating HaCaT cells compared with the cell rears. This asymmetry is most likely caused by a reduced cholesterol content of the lamellipodia as demonstrated by filipin staining. siRNA-mediated silencing of the cholesterol transporter ABCA1, which is known to redistribute cholesterol from rafts to non-raft regions, as well as pharmacological inhibition of this transporter with glibenclamide, strongly diminished the viscosity gradient of the plasma membrane. In addition, HaCaT cell migration was inhibited by glibenclamide treatment. These data suggest a preferential role of non-raft cholesterol in the establishment of the asymmetric plasma membrane viscosity.     

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.^1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.^4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.^6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the Förster Hoffman equation.⁹ This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.^6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.     

Precision assessment of biofluid viscosity measurements using molecular rotors

Blood viscosity changes with many pathologic conditions, but its importance has not been fully investigated because the current methods of measurement are poorly suited for clinical applications. The use of viscosity-sensitive fluorescent molecular rotors to determine fluid viscosity in a nonmechanical manner has been investigated recently, but it is unknown how the precision of the fluorescence-based method compares to established mechanical viscometry. Human blood plasma viscosity was modulated with high-viscosity plasma expanders, dextran, pentastarch, and hetastarch. The samples were divided into a calibration and a test set. The relationship between fluorescence emission and viscosity was established using the calibration set. Viscosity of the test set was determined by fluorescence and by cone-and-plate viscometer, and the precision of both methods compared. Molecular rotor fluorescence intensity showed a power law relationship with solution viscosity. Mechanical measurements deviated from the theoretical viscosity value by less than 7.6%, while fluorescence-based measurements deviated by less than 6%. The average coefficient of variation was 6.9% (mechanical measurement) and 3.4% to 3.8% (fluorescence-based measurement, depending on the molecular rotor used). Fluorescence-based viscometry exhibits comparable precision to mechanical viscometry. Fluorescence viscometry does not apply shear and is therefore more practical for biofluids which have apparent non-Newtonian properties. In addition, fluorescence instrumentation makes very fast serial measurements possible, thus promising new areas of application in laboratory and clinical settings.     

Partial characterization of six chlorophyll a-protein complexes isolated from a blue-green alga by a nondetergent method

Incorporation of cholesterol hemisuccinate into thylakoid membranes decreased the membrane fluidity as measured by polarized fluorescence from 1,6-diphenyl-1,3,5-hexatriene. Increasing membrane viscosity in this manner did not inhibit the thylakoid membrane protein kinase. In contrast the effects of the protein phosphorylation on State I-State II transitions, which were observed in untreated membranes, were abolished. This observation is interpreted as indicating that protein phosphorylation-induced energy transfer changes are sensitive to membrane viscosity because they might require a lateral migration of the light-harvesting complex serving Photosystem II from grana to stromal lamellae. Cation effects on room- and low-temperature fluorescence emission properties and membrane adhesion were not abolished in these cholesterol hemisuccinate-treated membranes.     

Role of mitochondrial membrane potential in the regulation of murine erythroleukemia cell differentiation

The level of cytoplasmic calcium ions appears to be important in the control of murine erythroleukemia (MEL) cell differentiation. Our interest in this study focuses on the relationship between the regulation of calcium concentration and differentiation. We used the fluorescent membrane probe DiOC₆ to examine the relationship between MEL cell mitochondria and changes in cytoplasmic calcium levels occurring at the initiation of commitment. Fluorescence microscopy reveals the selective association of DiOC₆ with MEL cell mitochondria, where an enhanced fluorescence is observed. Treatment of cells with dimethylsulfoxide (DMSO) or other inducers causes a decrease in mitochondria-associated fluorescence levels that occurs with the initiation of commitment. A decrease in DiOC₆ fluorescence is caused by agents that reduce mitochondrial membrane potential, but is only slightly affected by agents that alter plasma membrane potential. Amiloride and EGTA, agents that prevent commitment and inhibit calcium uptake, also prevent the decrease in DiOC₆ uptake caused by DMSO. The effect of DMSO on MEL cell mitochondria is mimicked by FCCP, a proton ionophore that dissipates mitochondrial membrane potential. FCCP also caused MEL cell mitochondria to release calcium into the cytoplasm. When MEL cells are treated with DMSO plus FCCP, commitment is initiated without the lag period observed when cells are treated with DMSO alone. These results are consistent with the hypothesis that mitochondrial transmembrane potential is important in the regulation of cytoplasmic calcium levels at the time of commitment of MEL cells to terminal differentiation.     

Effects of solvent polarity and solvent viscosity on the fluorescent properties of molecular rotors and related probes

Fluorescent molecular rotors belong to a group of twisted intramolecular charge transfer complexes (TICT) whose photophysical characteristics depend on their environment. In this study, the influence of solvent polarity and viscosity on several representative TICT compounds (three Coumarin derivatives, 4,4-dimethylaminobenzonitrile DMABN, 9-(dicyanovinyl)-julolidine DCVJ), was examined. While solvent polarity caused a bathochromic shift of peak emission in all compounds, this shift was lowest in the case of molecular rotors. Peak intensity was influenced strongly by solvent viscosity in DMABN and the molecular rotors, but polarity and viscosity influences cannot be separated with DMABN. Coumarins, on the other hand, did not show viscosity sensitivity. This study shows the unique suitability of molecular rotors as fluorescent viscosity sensors.     

A novel approach to blood plasma viscosity measurement using fluorescent molecular rotors

Molecular rotors, a group of fluorescent molecules with viscosity-dependent quantum yield, were tested for their suitability to act as fluorescence-based plasma viscometers. The viscosity of samples of human plasma was modified by the addition of pentastarch (molecular mass 260 kDa, 10% solution in saline) and measured with a Brookfield viscometer. Plasma viscosity was 1.6 mPa x s, and the mixtures ranged up to 4.5 mPa x s (21 degrees C). The stimulated light emission of the molecular rotors mixed in the plasma samples yielded light intensity that was nonoverlapping and of significantly different intensity for viscosity steps down to 0.3 mPa x s (n = 5, P < 0.0001). The mathematical relationship between intensity (I) and viscosity (eta) was found to be eta = (kappaI)(nu). After calibration and scaling the fluorescence based measurement had an average deviation versus the conventional viscometric measurements that was <1.8%. These results show the suitability of molecular rotors for fast, low-volume biofluid viscosity measurements achieving accuracy and precision comparable to mechanical viscometers.     

Luminescence Spectroscopy – a Useful Tool in Real-Time Monitoring of Viscosity during In-Vitro Digestion

Luminescence spectroscopy coupled with molecular rotors was used in the TNO Intestinal Model-1 (TIM-1) to monitor in situ changes to luminal viscosity of three maize starch samples varying in the amylose-to-amylopectin ratio (AM: AP): normal, high amylose (AM) and high amylopectin (AP). The fluorescence intensity (FI) of Fast Green (FG), a proven micro (and bulk) viscosity probe, was monitored throughout digestion to track changes in the gastric viscosity. The FI of FG and the viscosity imparted by the starch followed a power-law relationship. The emission of the MR was unaffected by the composition of TIM-1 secretion fluids nor pH. Hence, direct measurements of digesta FI are sensitive to changing viscosity during the simulated digestion. The viscosity was highest for AP, followed by normal starch, and high AM had the lowest viscosity. In the TIM-1 gastric compartment, from highest to lowest FI, and thus viscosity was high AM > high AP > normal maize starches. We conclude the validity of the proposed method to facilitate the measurement of luminal viscosity, in vitro, when the microviscosity represents bulk viscosity (i.e., when the increase in bulk viscosity is a result of molecular crowding and the surrounding environment around the rotor is homogeneous). Careful consideration is required when foods are heterogeneous as molecular rotors report only on their local non-uniform environment.

     

A molecular rotor as viscosity sensor in aqueous colloid solutions

BACKGROUND: Molecular rotors exhibit viscosity-dependent quantum yield, allowing non-mechanical determination of fluid viscosity. We analyzed fluorescence in the presence of viscosity-modulating macromolecules several orders of magnitude larger than the rotor molecule. METHOD OF APPROACH: Fluorescence of aqueous starch solutions with a molecular rotor in solution was related to viscosity obtained in a cone-and-plate viscometer. RESULTS: In dextran solutions, emission intensity was found to follow a power-law relationship with viscosity. Fluorescence in hydroxyethylstarch solutions showed biexponential behavior with different exponents at viscosities above and below 1.5 mPa s. Quantum yield was generally higher in hydroxyethylstarch than in dextran solutions. The power-law relationship was used to backcalculate viscosity from intensity with an average precision of 2.2% (range of -5.5% to 5.1%). CONCLUSIONS: This study indicates that hydrophilic molecular rotors are suitable as colloid solution viscosity probes after colloid-dependent calibration.     

Laurdan Fluorescence Lifetime Discriminates Cholesterol Content from?Changes in Fluidity in Living Cell Membranes

Detection of the fluorescent properties of Laurdan has been proven to be an efficient tool to investigate membrane packing and ordered lipid phases in model membranes and living cells. Traditionally the spectral shift of Laurdan’s emission from blue in the ordered lipid phase of the membrane (more rigid) toward green in the disordered lipid phase (more fluid) is quantified by the generalized polarization function. Here, we investigate the fluorescence lifetime of Laurdan at two different emission wavelengths and find that when the dipolar relaxation of Laurdan’s emission is spectrally isolated, analysis of the fluorescence decay can distinguish changes in membrane fluidity from changes in cholesterol content. Using the phasor representation to analyze changes in Laurdan’s fluorescence lifetime we obtain two different phasor trajectories for changes in polarity versus changes in cholesterol content. This gives us the ability to resolve in vivo membranes with different properties such as water content and cholesterol content and thus perform a more comprehensive analysis of cell membrane heterogeneity. We demonstrate this analysis in NIH3T3 cells using Laurdan as a biosensor to monitor changes in the membrane water content during cell migration.     

Environment-sensitive behavior of fluorescent molecular rotors

Molecular rotors are a group of fluorescent molecules that form twisted intramolecular charge transfer (TICT) states upon photoexcitation. When intramolecular twisting occurs, the molecular rotor returns to the ground state either by emission of a red-shifted emission band or by nonradiative relaxation. The emission properties are strongly solvent-dependent, and the solvent viscosity is the primary determinant of the fluorescent quantum yield from the planar (non-twisted) conformation. This viscosity-sensitive behavior gives rise to applications in, for example, fluid mechanics, polymer chemistry, cell physiology, and the food sciences. However, the relationship between bulk viscosity and the molecular-scale interaction of a molecular rotor with its environment are not fully understood. This review presents the pertinent theories of the rotor-solvent interaction on the molecular level and how this interaction leads to the viscosity-sensitive behavior. Furthermore, current applications of molecular rotors as microviscosity sensors are reviewed, and engineering aspects are presented on how measurement accuracy and precision can be improved.     

Comparison of excitation and emission ratiometric fluorescence methods for quantifying the membrane dipole potential

We are interested in developing fluorescence methods for quantifying lateral variations in the dipole potential across cell surfaces. Previous work in this laboratory showed that the ratio of fluorescence intensities of the voltage-sensitive dye di-8-ANEPPS using excitation wavelengths at 420 and 520 nm correlates well with measurements of the dipole potential. In the present work we evaluate the use of di-8-ANEPPS and an emission ratiometric method for measuring dipole potentials, as Bullen and Saggau (Biophys. J. 65 (1999) 2272-2287) have done to follow changes in the membrane potential in the presence of an externally applied field. Emission ratiometric methods have distinct advantages over excitation methods when applied to fluorescence microscopy because only a single wavelength is needed for excitation. We found that unlike the excitation ratio, the emission ratio does not correlate with the dipole potential of vesicles made from different lipids. A difference in the behaviour of the emission ratio in saturated compared to unsaturated lipid vesicles was noted. Furthermore, the emission ratio did not respond in the same way as the excitation ratio when cholesterol, 6-ketocholestanol, 7-ketocholesterol, and phloretin were added to dimyristoylphosphatidylcholine (DMPC) vesicles. We attribute the lack of correlation between the emission ratio and the dipole potential to simultaneous changes in membrane fluidity caused by changes in membrane composition, which do not occur when the electric field is externally applied as in the work of Bullen and Saggau. Di-8-ANEPPS can, thus, only be used via an excitation ratiometric method to quantify the dipole potential.     

Interaction of fluorescent molecular rotors with blood plasma proteins

Many disease states have associated blood viscosity changes. Molecular rotors, fluorescent molecules with viscosity sensitive quantum yields, have recently been investigated as a new method for biofluid viscosity measurement. Current viscometer measurements are complicated by proteins adhering to surfaces and forming air-surface layers. It is unknown at this time what effects proteins may have on biofluid viscosity measurements using molecular rotors. To answer this question, binding affinities to blood plasma proteins were investigated by equilibrium dialysis for four hydrophilic molecular rotors. Aqueous solutions of 9-[(2-cyano-2-hydroxy-carbonyl)vinyl]julolidine (CCVJ) and three derivatives were prepared and dialyzed against solutions of bovine source albumin, fibrinogen and immunoglobulin G approximating normal physiologic concentrations and fresh-frozen human plasma. After equilibration, dye concentration on each side of the dialysis membrane was assessed by spectrophotometry. The relative binding affinity of the four dyes to the proteins and to the plasma was compared. Affinity of all dyes was highest for albumin. The bound dye fraction showed little change in relation to protein concentration in the physiological concentration range. Diol, the most hydrophilic molecular rotor tested showed the lowest affinity for albumin. This study indicates that hydrophilic molecular rotors are well-suited for biofluid viscosity measurement.     

Hydrophilic molecular rotor derivatives-synthesis and characterization

Recent research shows high potential for some p-N,N-dialkylaminobenzylidenecyanoacetates, part of a group known as fluorescent molecular rotors, to serve as fluorescent, non-mechanical viscosity sensors. Of particular interest are molecules compatible with aqueous environments. In this study, we present the synthesis and physical characterization of derivatives from 9-(2-carboxy-2-cyanovinyl)-julolidine and related molecules. All compounds show a power-law relationship of fluorescence emission with the viscosity of the solvent, different mixtures of ethylene glycol and glycerol to modulate viscosity. Compounds with high water solubility exhibit the same behavior in aqueous solutions of dextran, where the dextran concentration was varied to modulate viscosity. In addition, some compounds have been found to have low sensitivity towards changes in the pH in the physiological range. The compounds presented show promise to be used in biofluids, such as blood plasma or lymphatic fluid, to rapidly and non-mechanically determine viscosity.     

Comparison of excitation and emission ratiometric fluorescence methods for quantifying the membrane dipole potential

We are interested in developing fluorescence methods for quantifying lateral variations in the dipole potential across cell surfaces. Previous work in this laboratory showed that the ratio of fluorescence intensities of the voltage-sensitive dye di-8-ANEPPS using excitation wavelengths at 420 and 520 nm correlates well with measurements of the dipole potential. In the present work we evaluate the use of di-8-ANEPPS and an emission ratiometric method for measuring dipole potentials, as Bullen and Saggau (Biophys. J. 65 (1999) 2272-2287) have done to follow changes in the membrane potential in the presence of an externally applied field. Emission ratiometric methods have distinct advantages over excitation methods when applied to fluorescence microscopy because only a single wavelength is needed for excitation. We found that unlike the excitation ratio, the emission ratio does not correlate with the dipole potential of vesicles made from different lipids. A difference in the behaviour of the emission ratio in saturated compared to unsaturated lipid vesicles was noted. Furthermore, the emission ratio did not respond in the same way as the excitation ratio when cholesterol, 6-ketocholestanol, 7-ketocholesterol, and phloretin were added to dimyristoylphosphatidylcholine (DMPC) vesicles. We attribute the lack of correlation between the emission ratio and the dipole potential to simultaneous changes in membrane fluidity caused by changes in membrane composition, which do not occur when the electric field is externally applied as in the work of Bullen and Saggau. Di-8-ANEPPS can, thus, only be used via an excitation ratiometric method to quantify the dipole potential.     

Cholesterol modulation of nicotinic acetylcholine receptor surface mobility

Nicotinic acetylcholine receptor (AChR) function and distribution are quite sensitive to cholesterol (Chol) levels in the plasma membrane (reviewed by Barrantes in J Neurochem 103 (suppl 1):72–80, 2007). Here we combined confocal fluorescence recovery after photobleaching (FRAP) and confocal fluorescence correlation spectroscopy (FCS) to examine the mobility of the AChR and its dependence on Chol content at the cell surface of a mammalian cell line. Plasma membrane AChR exhibited limited mobility and only ~55% of the fluorescence was recovered within 10 min after photobleaching. Depletion of membrane Chol by methyl-β-cyclodextrin strongly affected the mobility of the AChR at the plasma membrane; the fraction of mobile AChR fell from 55 to 20% in Chol-depleted cells, whereas Chol enrichment by methyl-β-cyclodextrin-Chol treatment did not reduce receptor mobility at the cell surface. Actin depolymerization caused by latrunculin A partially restored receptor mobility in Chol-depleted cells. In agreement with the FRAP data, scanning FCS experiments showed that the diffusion coefficient of the AChR was about 30% lower upon Chol depletion. Taken together, these results suggest that membrane Chol modulates AChR mobility at the plasma membrane through a Chol-dependent mechanism sensitive to cortical actin.