carbocyanines and 1-2-oleoylphosphatidylcholine

Entry of cell-penetrating peptides (CPPs) into living cells by translocating across plasma membranes is an important physiological phenomenon. To elucidate the mechanism of the translocation of CPPs across lipid bilayers, it is essential to reveal its elementary processes. For this purpose, here, we have developed a new method for the continuous, quantitative detection of the entry of CPPs into giant unilamellar vesicles (GUVs), where we investigate the interaction of fluorescent probe-labeled CPPs with single GUVs containing large unilamellar vesicles (LUVs) and fluorescent probes in their lumens using confocal microscopy. Using this method, we investigated the interaction of carboxyfluorescein (CF)-labeled transportan 10 (CF-TP10) with single GUVs comprised of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) containing LUVs of the same membrane and Alexa Fluor 647 hydrazide (AF647) in their lumens. At low concentrations of CF-TP10, first the fluorescence intensity (FI) of the GUV membrane increased with time, and then after some lag time the FI of the GUV lumen due to CF-TP10 increased continuously with time without leakage of AF647. At higher concentrations of CF-TP10, after the FI of the GUV lumen due to CF-TP10 increased significantly, leakage of AF647 started. These results indicate that CF-TP10 entered the GUV lumen by translocating across the GUV membrane and then bound to the LUVs there without pore formation and that CF-TP10 concentration in the lumen increased with time. The rate of entry of CF-TP10 into GUV lumen increased with CF-TP10 concentration. We discussed the kinetics of entry of CF-TP10 into single GUVs.

Topics: Carbocyanines; Cell-Penetrating Peptides; Fluoresceins; Fluorescent Dyes; Microscopy, Confocal; Phosphatidylcholines; Phosphatidylglycerols; Recombinant Fusion Proteins; Unilamellar Liposomes

To quantitatively examine the effect of membrane organization on lateral diffusion, we studied fluorescent carbocyanine lipid analogues and EGFP-tagged, single-pass transmembrane proteins in systems of decreasing complexity: (i) the plasma membrane (PM) of living cells, (ii) paraformaldehyde/dithiothreitol-induced giant plasma membrane vesicles (GPMVs), and (iii) giant unilamellar vesicles (GUVs) under physiological buffer conditions. A truncated, signaling-deficient interleukin-4 receptor subunit, showing efficient accumulation in the plasma membrane, served as a model transmembrane protein. Two-dimensional diffusion coefficients (D) were determined by fluorescence correlation spectroscopy (FCS) either at fixed positions (single-point, spFCS) or while scanning a circular orbit (circular scanning, csFCS). Consistent with a different inclusion sizes in the membrane, lipids diffuse slightly faster than the single-spanning membrane proteins in both membrane systems, GUVs and GPMVs. In GPMVs lipids and proteins consistently experienced a fivefold larger viscosity than in GUVs, reflecting the significant fraction of plasma membrane-derived proteins partitioning into GPMVs. Lipid and protein diffusion in the PM was, respectively, 2 times and 4-5 times slower in comparison to GPMVs. This discrepancy was quantitatively confirmed by csFCS. The similarity of diffusion of receptors and lipids in GPMVs and GUVs and its significant difference in the plasma membrane suggest that protein domains as small as EGFP convey sensitivity to the actin cortex on various length scales.

Topics: Carbocyanines; Cell Membrane; Cholesterol; Diffusion; Fluorescent Dyes; Gene Expression; Green Fluorescent Proteins; HEK293 Cells; Histidine; Humans; Interleukin-4; Interleukin-4 Receptor alpha Subunit; Kinetics; Oligopeptides; Phosphatidylcholines; Plasmids; Protein Transport; Recombinant Fusion Proteins; Spectrometry, Fluorescence; Sphingomyelins; Unilamellar Liposomes

The cell-penetrating peptide R9, an oligoarginine comprising nine arginines, has been used to transport biological cargos into cells. However, the mechanisms underlying its translocation across membranes remain unclear. In this report, we investigated the entry of carboxyfluorescein (CF)-labeled R9 (CF-R9) into single giant unilamellar vesicles (GUVs) of various lipid compositions and the CF-R9-induced leakage of a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using a method developed recently by us. First, we investigated the interaction of CF-R9 with dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) GUVs containing AF647 and small DOPG/DOPC vesicles. The fluorescence intensity of the GUV membrane due to CF-R9 (i.e., the rim intensity) increased with time to a steady-state value, and then the fluorescence intensity of the membranes of the small vesicles in the GUV lumen increased without leakage of AF647. This result indicates that CF-R9 entered the GUV lumen from the outside by translocating across the lipid membrane without forming pores through which AF647 could leak. The fraction of entry of CF-R9 at 6 min in the absence of pore formation, Pentry (6 min), increased with an increase in CF-R9 concentration, but the CF-R9 concentration in the lumen was low. We obtained similar results for dilauroyl-PG (DLPG)/ditridecanoyl-PC (DTPC) (2/8) GUVs. The values of Pentry (6 min) of CF-R9 for DLPG/DTPC (2/8) GUVs were larger than those obtained with DOPG/DOPC (2/8) GUVs at the same CF-R9 concentrations. In contrast, a high concentration of CF-R9 induced pores in DLPG/DTPC (4/6) GUVs through which CF-R9 entered the GUV lumen, so the CF-R9 concentration in the lumen was higher. However, CF-R9 could not enter DOPG/DOPC/cholesterol (2/6/4) GUVs. Analysis of the rim intensity showed that CF-R9 was located only in the outer monolayer of the DOPG/DOPC/cholesterol (2/6/4) GUVs. On the basis of analyses of these results, we discuss the elementary processes by which CF-R9 enters GUVs of various lipid compositions.

Topics: Biological Transport, Active; Carbocyanines; Cell-Penetrating Peptides; Cholesterol; Fluorescent Dyes; Membrane Lipids; Microscopy, Confocal; Oligopeptides; Phosphatidylcholines; Phosphatidylglycerols; Unilamellar Liposomes

Fluorescence correlation spectroscopy (FCS) measurements are widely used for determination of diffusion coefficients of lipids and proteins in biological membranes. In recent years, several variants of FCS have been introduced. However, a comprehensive comparison of these methods on identical systems has so far been lacking. In addition, there exist no consistent values of already determined diffusion coefficients for well-known or widely used membrane systems. This study aims to contribute to a better comparability of FCS experiments on membranes by determining the absolute diffusion coefficient of the fluorescent lipid analog 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine (DiD) in giant unilamellar vesicles (GUVs) made of dioleoylphosphatidylcholine (DOPC), which can in future studies be used as a reference value. For this purpose, five FCS variants, employing different calibration methods, were compared. Potential error sources for each particular FCS method and strategies to avoid them are discussed. The obtained absolute diffusion coefficients for DiD in DOPC were in good agreement for all investigated FCS variants. An average diffusion coefficient of D = 10.0 ± 0.4 μm(2) s(-1) at 23.5 ± 1.5 °C was obtained. The independent confirmation with different methods indicates that this value can be safely used for calibration purposes. Moreover, the comparability of the methods also in the case of slow diffusion was verified by measuring diffusion coefficients of DiD in GUVs consisting of DOPC and cholesterol.

Topics: Carbocyanines; Diffusion; Phosphatidylcholines; Spectrometry, Fluorescence

We report a fluorescence-based turn-on sensor for mapping the mechanical strain exerted by specific cell-surface proteins in living cells. The sensor generates force maps with high spatial and temporal resolution using conventional fluorescence microscopy. We demonstrate the approach by mapping mechanical forces during the early stages of regulatory endocytosis of the ligand-activated epidermal growth factor receptor (EGFR).

Topics: Biomechanical Phenomena; Biotin; Carbocyanines; Endocytosis; ErbB Receptors; Humans; Lipid Bilayers; Mechanoreceptors; Microscopy, Fluorescence; Nucleotides; Phosphatidylcholines; Phosphatidylethanolamines; Phosphorylation; Polyethylene Glycols; Rhodamines

Drug-membrane interactions are well known but poorly understood. Here we describe dual measurements of membrane thickness change and membrane area change due to the binding of the amphipathic drug curcumin. The combined results allowed us to analyze the binding states of a drug to lipid bilayers, one on the water-membrane interface and another in the hydrocarbon region of the bilayer. The transition between the two states is strongly affected by the elastic energy of membrane thinning (or, equivalently, area stretching) caused by interfacial binding. The data are well described by a two-state model including this elastic energy. The binding of curcumin follows a common pattern of amphipathic peptides binding to membranes, suggesting that the binding states of curcumin are typical for amphipathic drugs.

Topics: Binding Sites; Carbocyanines; Curcumin; Dimethyl Sulfoxide; Lipid Bilayers; Membrane Fluidity; Models, Chemical; Phosphatidylcholines; Phosphatidylethanolamines; Rhodamines; Unilamellar Liposomes

The behavior of the cationic hemicyanines trans-4-[4-(dimethylamino)-styryl]-1-methylpyridinium iodide (HC) and 4,(4-(dihexadecylamino)styryl-N-methyl-pyridinium iodide (DIA) were studied in large unilamellar vesicles (LUV) of 1,2-di-oleoyl-sn-glycero-3-phosphatidylcholine (DOPC) using absorption, emission, depolarization and time resolved spectroscopies. Also, thorough spectroscopic studies were performed in homogeneous media to investigate the different interactions that the dyes can experience with its microenvironment. These results help us to comprehend the dye performance under different media and, consequently find interesting features of the DOPC membrane properties. The studies in homogeneous media analyzed by the Kamlet and Taft's solvatochromic comparison method demonstrate, for the first time, that the cationic hemycianines undergo specific interactions with the medium through the solvents ability to donate an electron pair as measured by the beta parameter. Thus, the absorption bands shifts bathochromically with beta while, the emission band shifts hypsochromically. In addition, for the relaxed hemicyanines the 00 energy, nu00, is invariant with the solvent properties. The results in LUV of DOPC show that, DIA undergoes a strong association with the vesicle bilayer while HC partitions between the water and the bilayer pseudophases. To monitor directly the microenvironment and dynamics around HC and DIA inside the DOPC bilayer, we use the wavelength-selective fluorescence approach, which is based on the red edge effect in fluorescence spectroscopy, in addition with the nu00 energy of the hemicyanines. The results show that the fluid state of the DOPC bilayer resembles the microenvironment of sodium bis (2-ethylhexyl) sulfosuccinate (AOT) reverse micelles at W=[H2O]/[AOT] below 10 where there is no free water forming the water pool. Moreover, it is demonstrated for the first time, that the region of the bilayer close to the polar head of DOPC is a powerful electron donor environment.

Topics: Carbocyanines; Cations; Fluorescence; Fluorescent Dyes; Lipid Bilayers; Molecular Structure; Optics and Photonics; Phosphatidylcholines; Pyridinium Compounds

Upon cold and drought stress, sucrose and trehalose protect membrane structures from fusion and leakage. Similarly, these sugars protect membrane proteins from inactivation during dehydration. We studied the interactions between sugars and phospholipid membranes in giant unilamellar vesicles with the fluorescent lipid analog 3,3'-dioctadecyloxacarbocyanine perchlorate incorporated. Using fluorescence correlation spectroscopy, it was found that sucrose decreased the lateral mobility of phospholipids in the fully rehydrated, liquid crystalline membrane more than other sugars did, including trehalose. To describe the nature of the difference in the interaction of phospholipids with sucrose and trehalose, atomistic molecular dynamics studies were performed. Simulations up to 100 ns showed that sucrose interacted with more phospholipid headgroups simultaneously than trehalose, resulting in a larger decrease of the lateral mobility. Using coarse-grained molecular dynamics, we show that this increase in interactions can lead to a relatively large decrease in lateral phospholipid mobility.

Topics: Carbocyanines; Fluorescent Dyes; Membrane Fluidity; Membranes, Artificial; Phosphatidylcholines; Spectrometry, Fluorescence; Sucrose; Trehalose

The influence of cholesterol on the sphingomyelin (SM)/dioleoylphosphatidylcholine (DOPC) binary system was investigated in various respects. Electron spin resonance (ESR) measurements reveal that the order parameter of 5DS (5-doxyl stearic acid) in SM/DOPC bilayers increases notably when the concentration of cholesterol is over 30 mol%. Membrane potential measurements indicate that the K+ permeability of the SM/DOPC bilayer decreases steeply at 40 mol% cholesterol concentration. Both these experiments suggest that cholesterol reduces the motion amplitude of hydrocarbon chains abruptly above 30 mol%. In contrast to the ordering effects on the hydrocarbon chains, (31)P-NMR results indicate that cholesterol slightly increases the motion of phosphate groups of the lipids. (31)P-NMR also raises the possibility of domain formation in the presence of cholesterol. Fluorescence-quenching experiments verified that solid domains appear in the binary system when cholesterol is present, and percolation threshold occurs at 50 mol% cholesterol concentration. The solid domains bear the properties of liquid ordered phase, which is the basic structure of caveolae and functional rafts. So this work provides an artificial model for the study of rafts and caveolae on biological membranes.

Topics: Animals; Benzothiazoles; Biophysical Phenomena; Biophysics; Carbocyanines; Cholesterol; Egg Yolk; Electron Spin Resonance Spectroscopy; Fluorescence; Lipid Bilayers; Magnetic Resonance Spectroscopy; Membrane Lipids; Membrane Potentials; Membranes, Artificial; Phosphatidylcholines; Sphingomyelins; Spin Labels; Temperature; Time Factors

We show that the lipophilic, cationic fluorescent dyes R18 and Dil translocate from one monolayer of a phospholipid bilayer membrane to the other in a concentration and voltage-dependent manner. When the probes were incorporated into voltage-clamped planar membranes and potentials were applied, displacement currents resulted. The charged probes sensed a large fraction of the applied field. When these probes were added to only one monolayer, displacement currents were symmetrical around 0 mV, indicating that the probes distributed equally between the two monolayers. Charge translocation required that the bilayer be fluid. When membranes were in a condensed gel phase, displacement currents were not observed; raising the temperature to above the gel-liquid crystalline transition restored the currents. Translocation of R18 was also shown by fluorescence measurements. When R18 was in the bilayer at high, self-quenching concentrations, voltage pulses led to voltage-dependent fluorescence changes. The kinetics of the fluorescence changes and charge translocations correlated. Adding the quencher I- to one aqueous phase caused fluorescence to decrease or increase when voltage moved R18 toward or away from the quencher at low, nonquenching concentrations of R18. In contrast to R18, Dil incorporated into bilayers was a carrier fo I-, and hence I- altered Dil currents. Voltage-driven translocations allow R18 and Dil to be used to probe membrane potential changes.

Topics: Carbocyanines; Fluorescent Dyes; Iodides; Lipid Bilayers; Membrane Lipids; Membrane Potentials; Membranes, Artificial; Phosphatidylcholines; Rhodamines; Solubility; Spectrometry, Fluorescence

Monolayers of dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and some mixtures of these lipids were investigated using an epifluorescence microscopic surface balance. Monolayers were visualized at 23 +/- 1 degree C through the fluorescence of 1 mol% of two different fluorescent probes, 1-palmitoyl-2-(12-[(7-nitro-2-1,3-benzoxadizole-4- yl)amino]dodecanoyl)phosphatidylcholine (NBD-PC), which partitions into the liquid expanded (LE) or disordered lipid phase and 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO-C18), which preferentially associates with the liquid condensed (LC) phase or lipid with ordered chains. LC domains were observed in pure DPPC monolayers at relatively low surface pressures (pi), and these domains grew with increasing surface pressure. Only liquid expanded phase was observed in pure DOPC monolayers up to the point of monolayer collapse. In monolayers containing 29:70:1, 49:50:1, and 69:30:1 (mol/mol/mol) of DPPC:DOPC:probe the domains of LC phase were smaller than those seen in DPPC monolayers at equivalent surface pressures. Quantitative analysis of the visual fields shown by the mixed monolayers showed a distribution of sizes of condensed domains at any given pi. At pi = 30 mN m-1, liquid-expanded, or fluid, regions occupied more than 70% of the total monolayer area in all three mixtures studied, whereas DPPC monolayers were more than 75% condensed or solid at that pressure. For monolayers of DPPC:DOPC:NBD-PC 49:50:1 and 69:30:1 the average domain size and the percentage of the total area covered with LC, or rigid, areas increased to a maximum at pi around 35 mN m-1 followed by a decrease at higher pi. Repetitive compression and expansion of the monolayers containing DPPC:DOPC:NBD-PC 49:50:1 at an initial rate of 3.2 A2 molecule-1 s-1 produced monolayers with visual properties consistent with there being a preferential exclusion of the unsaturated lipid from the monolayer.

Topics: 1,2-Dipalmitoylphosphatidylcholine; 4-Chloro-7-nitrobenzofurazan; Biophysical Phenomena; Biophysics; Carbocyanines; Fluorescent Dyes; Membranes, Artificial; Microscopy, Fluorescence; Phosphatidylcholines; Pulmonary Surfactants

In this study the effect of a transmembrane electrical potential on the phospholipid headgroup conformation was investigated using the 2H-NMR technique. Large unilamellar vesicles were prepared of dioleoylphosphatidylcholine, specifically 2H-labeled at the alpha- or beta-position of the choline group. No conformational change of the phosphocholine headgroup could be detected after induction of a valinomycin-induced K(+)-diffusion potential across the bilayer. However, this method could be used to measure the redistribution of tetraphenylphosphonium across the bilayer in response to delta psi, which reorients the phosphocholine headgroups in the opposite bilayer-water interfaces.

Topics: Benzothiazoles; Carbocyanines; Coloring Agents; Deuterium; Lipid Bilayers; Liposomes; Magnetic Resonance Spectroscopy; Membrane Potentials; Molecular Conformation; Onium Compounds; Organophosphorus Compounds; Phosphatidylcholines; Phosphatidylglycerols; Phosphorylcholine; Tetraphenylborate