Effect of lateral mobility of fluorescent probes in lipid mixing assays of cell fusion.

Monolayers of human erythrocytes, immobilized on a cover slip, were induced to fuse by polyethylene glycol (mol wt 8,000). The mobility of fluorescent probes, 1-oleoyl-2-[12-[(7-nitro-2,1,3-benzoxadizol-4-yl)amino]dodecanoyl] phosphatidyl-choline (C12-NBD-PC), from labeled cells to unlabeled cells was monitored by video-enhanced fluorescence microscopy. A dequenching curve was obtained from the measurement of fluorescence intensities of pairs of fused cells over time. The dequenching curve and the curve obtained from macroscopic measurements of a cell monolayer (described in the preceding article) were compared and discussed. The slow probe transfer rate between a pair of fused cells was explained by a diffusion model based on membrane area conservation and the geometry of the fusion lumen. An equivalent lumen between two fused cells, thought to be the main rate limitation of probe mobility after fusion, was calculated to be approximately 130 nm in diameter. Lumens of 75 nm in diameter were observed by electron microscopy. Thus, the rate of macroscopic fluorescence dequenching depends not only upon the fusion efficiency, but also upon the number of simultaneous fusion partners, the geometry of their contact points, and the lateral mobility of the fluorescent probes through these points. The relative fusion efficiency can be derived only from the saturation dequenching values.     

Partitioning of exchangeable fluorescent phospholipids and sphingolipids between different lipid bilayer environments

Exchangeable phospho- and sphingolipid probes (phosphatidylcholine, -ethanolamine, -serine, and -glycerol, phosphatidic acid, sphingomyelin, cerebroside, and sulfatide) have been synthesized in which one acyl chain is substituted with a fluorescent bimanyl, 7-(dimethylamino)coumarin-3-yl, or diphenyl-hexatrienyl group. The distribution of these probes between two different populations of lipid vesicles can be readily monitored by fluorescence intensity measurements, as described by Nichols and Pagano [Nichols, J. W., & Pagano, R. E. (1982) Biochemistry 21, 1720-1726], when one of the vesicle populations contains a low mole fraction of a nonexchangeable quencher, (12-DABS)-18-PC. The probes examined in this study exchange between phospholipid vesicles on a time scale of minutes, with kinetics indicating that the transfer process takes place by diffusion of probe monomers through the aqueous phase. As expected, lipid probes with different charges differ markedly in their equilibrium distributions between neutral and charged lipid vesicles. However, probes with different polar headgroups differ only modestly in their relative affinities for vesicles composed of "hydrogen-bonding" lipids (PE and PS) vs "non-hydrogen-bonding" lipids (PC and PG or O-methyl-PA). Probes with different headgroups also show modest, albeit reproducible, differences in their relative affinities for cholesterol-containing vs cholesterol-free PC/PG vesicles. Our results suggest that lipids with different headgroup structures may mix more nearly ideally in liquid-crystalline lipid bilayers than would be predicted from previous analyses of the phase diagrams for binary lipid mixtures.     

Interactions of Synphilin-1 with phospholipids and lipid membranes

Synphilin-1 is an alpha-synuclein binding protein that is involved in the pathogenesis of Parkinson's disease. The present study investigated the phospholipid-binding capacity of Synphilin-1. The C-terminus of Synphilin-1 was found to selectively bind to acidic phospholipids, including phosphatidic acid, phosphatidylserine, and phosphatidylglycerol, but not to naturally charged phospholipids. Synphilin-1 was targeted to cytoplasmic lipid droplets in mammalian cells. The amino acid sequence 610-640 was found to represent the primary determinant site for phospholipid binding. Moreover, the R621C mutation identified in Parkinson's disease abolished Synphilin-1 association with lipid droplets. The lipophilicity of Synphilin-1 might prove relevant to its physiologic function.     

Novel fluorescent technology platform for high throughput cytotoxicity and proliferation assays

We have developed a novel fluorescent Oxygen BioSensor technology platform adaptable to many applications in the area of drug discovery and development, particularly cell-based assays. This biosensor technology requires no additional reagents or incubations, and affords continuous real-time readout of dissolved oxygen concentrations. Since the level of oxygen dissolved in an assay's medium correlates to the number and viability of the cells in the medium, this technology is ideally suited for monitoring cell viability, proliferation, or death. The technology is particularly well suited to investigating cells' kinetic responses to proliferative or toxic stimuli, such as drugs. When incorporated into a 96- or 384-well microplate format, it is compatible with standard laboratory automation systems. Here we present data illustrating the application of the Oxygen BioSensor technology for rapid, homogeneous detection and evaluation of metabolic activity of a variety of eukaryotic and prokaryotic cells, including mammalian cells, insect cells, yeast, and bacteria. In the absence of toxic substances, we find a good correlation between cell number and signal over a wide range of cell concentrations and growth times. To evaluate the usefulness of the Oxygen BioSensor for cytotoxicity assays, we have performed a series of experiments using a range of toxic agents and cell types, including both bacteria and mammalian cell lines. In a side-by-side comparison to standard MTT assays using HL60 cells, comparable IC(50) values were found with the Oxygen BioSensor for five different toxins or drugs. This assay method does not have the need for additional reagents, handling steps, or incubation periods required by the MTT assays.     

Rotation of fluorescent probes localized within lipid bilayer membranes

Measurements of the steady state polarization of fluorescence from perylene and 9-vinylanthracene embedded in bilayer membranes were performed as a function of temperature. Similar measurements were made when these probes were dissolved in hydrocarbons as model solvents. The effects of cholesterol and n-alkyl alcohol additions to bilayers and head group variation were also examined. Results were expressed in terms of the average rotation rates of the probes.At 25°C, the calculated rotation rate for perylene in egg phosphatidylcholine vesicles was 275 × 10⁶ sec^?1 as compared to 2400 × 10⁶ sec^?1 for perylene in n-hexadecane. However, the activation energies for probe rotation in both environments was about 7 kcal/mole suggesting similar rotational diffusion mechanisms. Membrane microviscosity evaluations were performed according to a recently published scheme and an assessment of this method of viscosity estimation was given. The presence of an approximately equimolar amount of cholesterol impeded probe rotation (90 × 10⁶ sec^?1 at 25°C) and reduced the activation energy (4.9 kcal/mole) for probe rotation. In contrast, addition of n-alkyl alcohols to the vesicle suspension acted to increase probe rotation rates, an indication of fluidization of the membranes. This is in accord with spin label and cation permeability data for similar membranes.It was concluded that this method of probing can adequately report changes in membrane dynamic structure when these changes occur uniformly over the membrane surface. The interpretation is less clear when structural changes occur only in patches or domains of the membrane thereby producing a non-uniform surface distribution of probes.     

Exchange of phospholipids between brain membranes in vitro

1. When unlabelled mitochondria from guinea-pig brain were incubated with a (32)P-labelled microsomal fraction from brain there was a transfer of phospholipid to the mitochondria, which could not be accounted for by an aggregation of microsomes and mitochondria or an exchange with microsomes contaminating the mitochondria. Under similar circumstances there was a transfer of phospholipid from (32)P-labelled mitochondria to microsomes, indicating that the process was one of exchange. 2. The transfer from microsomes was greatly stimulated by a non-dialysable heat-labile macromolecular component in the brain supernatant fraction but not by the concentration of the particulate fractions. 3. Phospholipid-exchange processes occurred most readily between pH7 and 7.5 and were inhibited by the presence of myelin and on the addition of lysophosphatidylcholine. 4. The rates of transfer of individual phospholipids from brain microsomes to mitochondria were similar. 5. (32)P-labelled microsomes could slowly donate phospholipid to the isolated synaptosomal (nerve-ending) fraction but the phospholipids of the myelin fraction did not exchange. 6. Subfractionation of the synaptosomal fraction after [(32)P]phospholipid transfer showed that the mitochondria were most actively labelled during the incubation. All of the isolated individual synaptosomal membranes were capable of acquiring phospholipid on incubation with a (32)P-labelled brain supernatant fraction although a greater percentage was again exchanged by the mitochondrial fraction.     

Measuring translocation of fluorescent lipid derivatives across yeast Golgi membranes

Phospholipid asymmetry is a fundamental feature of the plasma membrane of most eukaryotic cells and its regulation is linked to diverse physiological processes such as apoptosis and blood clotting [P. Williamson, R.A. Schlegel, Biochim. Biophys. Acta 1585 (2002) 53-63; R.F. Zwaal, A.J. Schroit, Blood 89 (1997) 1121-1132]. In addition, the phospholipid translocases (flippases) that are thought to establish asymmetry are also implicated in vesicle-mediated protein transport throughout the secretory and endocytic pathways [T.R. Graham, Trends Cell Biol. 14 (2004) 670-677]. However, the biochemical properties of phospholipid translocases in membranes of the Golgi complex and endosomes have received much less attention than translocases in the plasma membrane. We describe here a method for purifying yeast Golgi membranes and assaying an ATP-dependent phospholipid translocase activity in these membranes using fluorescent lipid analogues. This assay detects ATP-dependent translocation of labeled phosphatidylserine across late Golgi membranes, which requires the activity of a P-type ATPase called Drs2p [P. Natarajan, J. Wang, Z. Hua, T.R. Graham, Proc. Natl. Acad. Sci. USA 101 (2004) 10614-10619].     

Transbilayer movement of phospholipids at the main phase transition of lipid membranes: implications for rapid flip-flop in biological membranes

The transbilayer movement of fluorescent phospholipid analogs in liposomes was studied at the lipid phase transition of phospholipid membranes. Two NBD-labeled analogs were used, one bearing the fluorescent moiety at a short fatty acid chain in the sn-2 position (C(6)-NBD-PC) and one headgroup-labeled analog having two long fatty acyl chains (N-NBD-PE). The transbilayer redistribution of the analogs was assessed by a dithionite-based assay. We observed a drastic increase of the transbilayer movement of both analogs at the lipid phase transition of DPPC (T(c) = 41 degrees C) and DMPC (T(c) = 23 degrees C). The flip-flop of analogs was fast at the T(c) of DPPC with a half-time (t(1/2)) of ~6-10 min and even faster at the T(c) of DMPC with t(1/2) on the order of <2 min, as shown for C(6)-NBD-PC. Suppressing the phase transition by the addition of cholesterol, the rapid transbilayer movement was abolished. Molecular packing defects at the phase transition are assumed to be responsible for the rapid transbilayer movement. The relevance of those defects for understanding of the activity of flippases is discussed.     

Rapid transbilayer movement of the fluorescent sterol dehydroergosterol in lipid membranes

This study establishes a new assay for measuring the transbilayer movement of dehydroergosterol (DHE) in lipid membranes. The assay is based on the rapid extraction of DHE by methyl-beta-cyclodextrin (M-CD) from liposomes. The concentration of DHE in the liposomal membrane was measured by using fluorescence resonance energy transfer (FRET) from DHE to dansyl-phosphatidylethanolamine, which is not extracted from liposomes by M-CD. The method was applied to small (SUV) and large (LUV) unilamellar vesicles of different compositions and at various temperatures. From the kinetics of FRET changes upon extraction of DHE from membranes, rates of M-CD mediated extraction and flip-flop of DHE could be deduced and were found to be dependent on the physical state of the lipid phase. For egg phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in the liquid-crystalline state, halftimes of extraction and transbilayer movement were <5 s and approximately 20-50 s, respectively, at 10 degrees C. For 1,2-dimyristoyl-sn-glycero-3-phosphocholine-SUV being in the gel state at 10 degrees C, the respective halftimes were 28 s and 5-8 min. Surprisingly, DHE could not be extracted from LUV consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. This might be an indication of specific interactions between DHE molecules in membranes depending on the phospholipid composition of the membrane.     

Novel assays for proteases using green fluorescent protein-tagged substrate immobilized on a membrane disk

Green fluorescent protein (GFP) is very stable for various proteases. Using this property, three protease assay methods designated the disk separation assay for remaining GFP (DSAR), the disk separation assay for liberated GFP (DSAL), and the homogeneous assay for fluorescence concentrated on membrane (HAFCOM) were developed. These methods employ a nylon membrane designated “Cleave-Checker” on which GFP-SpA_B (domain B in staphylococcal protein A) is immobilized. The SpA_B region was used as a substrate for the protease, and the isolation of GFP from the membrane generated by the digestion of the SpA_B region was detected. In DSAR, it was possible to detect solution of at least 25 ng/ml trypsin or proteinase K by visual observation. The most important feature of DSAR is that the detection of the protease is possible only under UV light. In contrast, DSAL is suitable for a highly sensitive assay. The assay ranges of DSAL were 1.6 to 100 ng/ml in trypsin and 1.6 to 400 ng/ml in proteinase K. HAFCOM does not require bound/free (B/F) separation; thus, the procedure is simpler than that with DSAL and the reproducibility is high. The assay ranges of HAFCOM were 25 to 400 ng/ml in trypsin and 12.5 to 200 ng/ml in proteinase K. The Cleave-Checker used for these methods was stable in a dry state, and long-term preservation for at least several months was possible.     

The effect of hydroxylated fatty acid-containing phospholipids in the remodeling of lipid membranes

The synthetic fatty acid 2-hydroxyoleic acid (2OHOA) is an antitumor drug that regulates membrane lipid composition and structure. An important effect of this drug is the restoration of sphingomyelin (SM) levels in cancer cell membranes, where the SM concentration is lower than in non-tumor cells. It is well known that free fatty acid concentration in cell membranes is lower than 5%, and that fatty acid excess is rapidly incorporated into phospholipids. In a recent work, we have considered the effect of free 2OHOA in model membranes in liquid ordered (Lo) and liquid disordered (Ld) phases, by using all-atom molecular dynamics. This study concerns membranes that are modified upon incorporation of 2OHOA into different phospholipids. 2OHOA-containing phospholipids have a permanent effect on lipid membranes, making a Ld membrane surface more compact and less hydrated, whereas the opposite effect is observed in Lo domains. Moreover, the hydroxyl group of fatty acid chains increases the propensity of Ld model membranes to form hexagonal or other non-lamellar structures. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Structural and dynamic effects of oxidatively modified phospholipids in unsaturated lipid membranes.

Phospholipid hydroperoxides and phospholipid alcohols are two of the major forms of oxidatively modified phospholipids produced during oxidant stress and lipid peroxidation. The process of lipid peroxidation is known to affect the physiological function of membranes. We, therefore, investigated the effects of lipid peroxidation products on the molecular interactions in membranes. Our study was specifically focused on the effects of lipid peroxidation products on static membrane structure (molecular orientational order) and on the reorientational dynamics of the probe molecules in lipid bilayers. The study was done by performing angle-resolved fluorescence depolarization measurements (AFD) on the fluorescent probe diphenylhexatriene (DPH) and by performing angle-resolved electron spin resonance (A-ESR) measurements on cholestane (CSL) nitroxide spin probes embedded in macroscopically oriented planar bilayers consisting of 2-10% 1-palmitoyl-2-(9/13-hydroperoxylinoleoyl)phosphatidylcholine (PLPC-OOH) or 1-palmitoyl-2-(9/13-hydroxylinoleoyl)phosphatidylcholine (PLPC-OH) in 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC) or dilinoleoylphosphatidylcholine (DLPC). Both probe molecules have rigid cylindrical geometries and report on the overall molecular order and dynamics. However, being more polar, the nitroxide spin probe CSL is preferentially located near the surface of the membrane, while the less polar fluorescent probe DPH reports preferentially near the central hydrophobic region of the lipid bilayers. The results show that the presence of relatively small amounts of oxidatively modified phospholipids within the PLPC or DLPC membranes causes pronounced structural effects as the molecular orientational order of the probe molecules is strongly decreased. In contrast, the effect on membrane reorientational dynamics is minimal.     

Cholesterol and anionic phospholipids increase the binding of amyloidogenic transthyretin to lipid membranes

Deposition of transthyretin (TTR) amyloid is a pathological hallmark of familial amyloidotic polyneuropathy (FAP). Recently we showed that TTR binds to membrane lipids via electrostatic interactions and that membrane binding is correlated with the cytotoxicity induced by amyloidogenic TTR. In the present study, we examined the role of lipid composition in membrane binding of TTR by a surface plasmon resonance (SPR) approach. TTR bound to lipid bilayers through both high- and low-affinity interactions. Increasing the mole fraction of cholesterol in the bilayer led to an increase in the amount of high-affinity binding of an amyloidogenic mutant (L55P) TTR. In addition, a greater amount of L55P TTR bound with high affinity to membranes made from anionic phospholipids, phosphatidylglycerol (PG) and phosphatidylserine (PS), than to membranes made from zwitterionic phospholipid phosphatidylcholine (PC). The anionic phospholipids (PS and PG) promoted the aggregation of L55P TTR by accelerating the nucleation phase of aggregation, whereas the zwitterionic phospholipid PC had little effect. These results suggest that cholesterol and anionic phospholipids may be important for TTR aggregation and TTR-induced cytotoxicity.     

Cholesterol and anionic phospholipids increase the binding of amyloidogenic transthyretin to lipid membranes

Deposition of transthyretin (TTR) amyloid is a pathological hallmark of familial amyloidotic polyneuropathy (FAP). Recently we showed that TTR binds to membrane lipids via electrostatic interactions and that membrane binding is correlated with the cytotoxicity induced by amyloidogenic TTR. In the present study, we examined the role of lipid composition in membrane binding of TTR by a surface plasmon resonance (SPR) approach. TTR bound to lipid bilayers through both high- and low-affinity interactions. Increasing the mole fraction of cholesterol in the bilayer led to an increase in the amount of high-affinity binding of an amyloidogenic mutant (L55P) TTR. In addition, a greater amount of L55P TTR bound with high affinity to membranes made from anionic phospholipids, phosphatidylglycerol (PG) and phosphatidylserine (PS), than to membranes made from zwitterionic phospholipid phosphatidylcholine (PC). The anionic phospholipids (PS and PG) promoted the aggregation of L55P TTR by accelerating the nucleation phase of aggregation, whereas the zwitterionic phospholipid PC had little effect. These results suggest that cholesterol and anionic phospholipids may be important for TTR aggregation and TTR-induced cytotoxicity.     

Binding of a fluorescent lipid amphiphile to albumin and its transfer to lipid bilayer membranes

Kinetics and thermodynamics of the binding of a fluorescent lipid amphiphile, Rhodamine Green(TM)-tetradecylamide (RG-C(14:0)), to bovine serum albumin were characterized in an equilibrium titration and by stopped-flow fluorimetry. The binding equilibrium of RG-C(14:0) to albumin was then used to reduce its concentration in the aqueous phase to a value below its critical micelle concentration. Under these conditions, the only two species of RG-C(14:0) in the system were the monomer in aqueous solution in equilibrium with the protein-bound species. After previous determination of the kinetic and thermodynamic parameters for association of RG-C(14:0) with albumin, the kinetics of insertion of the amphiphile into and desorption off lipid bilayer membranes in different phases (solid, liquid-ordered, and liquid-disordered phases, presented as large unilamellar vesicles) were studied by stopped-flow fluorimetry at 30 degrees C. Insertion and desorption rate constants for association of the RG-C(14:0) monomer with the lipid bilayers were used to obtain lipid/water equilibrium partition coefficients for this fluorescent amphiphile. The direct measurement of these partition coefficients is shown to provide a new method for the indirect determination of the equilibrium partition coefficient of similar molecules between two defined lipid phases if they coexist in the same membrane.     

Selective association of phospholipids as a clue for the passive flip-flop diffusion through bilayer lipid membranes

We showed that the investigation of the selective association of phospholipids might contribute to the insight of the flip-flop diffusion processes. The process of selective association was studied quantitatively by testing the association probabilities for both parallel and anti-parallel orientations of the polar headgroups. The model of double chain binary mixture confirms a high capacity of phospholipids for self-association in parallel configuration of the electric dipole moments whether the cross-sectional area of the polar headgroups are in an usual range of 25–55 Ų. It is demonstrated that the aggregation of a class of phospholipids from a binary mixture is strongly dependent on the dipole-dipole interaction between the same phospholipids and is modulated by the magnitude of the electric dipole moment of the other phospholipids from that binary mixture. There are a great number of mechanisms involved in the transbilayer movement of phospholipids. We referred here only to the passive transport of lipids from one monolayer to the other. The flip-flop mechanisms raised in this paper are the breakdown of bilayer due to the increase of the packing density and the inversion of the coupled phospholipids from the opposite monolayers of the same bilayer. Thus, the pair formation promoting a drop in occupied volume decreases the packing pressure in the respective monolayer and consequently triggers a flip-flop into the other direction since the packing pressure in the other monolayer has not dropped. According to the present model for the binary mixtures of double-chain lipids, the rate of the flip-flop diffusion decreased by increasing the number of the methylene groups added to the acyl chain. This dependence may be perturbed whether the phospholipids possesses a very high cross-section area of the polar headgroups (a > 55 Ų). We think that the selective association of phospholipids is neither exclusively, nor only involved in promoting the transbilayer diffusion of phospholipids. Most probably, the selective association determines some phospholipid domains that attract certain particular proteins so that it can modulate the protein activity.