Lateral diffusion and fluorescence microscope studies on a monoclonal antibody specifically bound to supported phospholipid bilayers

Supported phospholipid bilayers prepared by Langmuir-Blodgett techniques were introduced recently as a new model membrane system [Tamm, L.K., & McConnell, H.M. (1985) Biophys. J. 47, 105-113]. Here, supported bilayers are applied to study the lateral diffusion and lateral distribution of membrane-bound monoclonal antibodies. A monoclonal anti-trinitrophenol antibody was found to bind strongly and with high specificity to supported phospholipid bilayers containing the lipid hapten (trinitrophenyl)phosphatidylethanolamine at various mole fractions. The lateral distribution of the membrane-bound antibodies was studied by epifluorescence microscopy. The bound antibodies aggregated into patches on a host lipid bilayer of dimyristoylphosphatidylcholine below the lipid chain melting phase transition and redistributed uniformly on fluid-phase supported bilayers. Lateral diffusion coefficients and mobile fractions of fluorescent phospholipid analogues and fluorescein-labeled antibodies were measured by fluorescence recovery after pattern photobleaching. The lateral diffusion coefficients of the membrane-bound antibodies resembled those of the phospholipids but were reduced by a factor of 2 in the fluid phase. The lipid chain melting phase transition was also reflected in the lateral diffusion coefficient of the bound antibody but occurred at a temperature about 3 deg higher than the phase transition in supported bilayers of pure phospholipids. The antibody lateral diffusion coefficients decreased in titration experiments monotonically with increasing antibody surface concentrations by a factor of 2-3. Correspondingly, a relatively small decrease of the antibody lateral diffusion coefficient was observed with increasing mole fractions of lipid haptens in the supported bilayer.     

Tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: silane-polyethyleneglycol-lipid as a cushion and covalent linker

There is increasing interest in supported membranes as models of biological membranes and as a physiological matrix for studying the structure and function of membrane proteins and receptors. A common problem of protein-lipid bilayers that are directly supported on a hydrophilic substrate is nonphysiological interactions of integral membrane proteins with the solid support to the extent that they will not diffuse in the plane of the membrane. To alleviate some of these problems we have developed a new tethered polymer-supported planar lipid bilayer system, which permitted us to reconstitute integral membrane proteins in a laterally mobile form. We have supported lipid bilayers on a newly designed polyethyleneglycol cushion, which provided a soft support and, for increased stability, covalent linkage of the membranes to the supporting quartz or glass substrates. The formation and morphology of the bilayers were followed by total internal reflection and epifluorescence microscopy, and the lateral diffusion of the lipids and proteins in the bilayer was monitored by fluorescence recovery after photobleaching. Uniform bilayers with high lateral lipid diffusion coefficients (0.8-1.2 x 10(-8) cm(2)/s) were observed when the polymer concentration was kept slightly below the mushroom-to-brush transition. Cytochrome b(5) and annexin V were used as first test proteins in this system. When reconstituted in supported bilayers that were directly supported on quartz, both proteins were largely immobile with mobile fractions < 25%. However, two populations of laterally mobile proteins were observed in the polymer-supported bilayers. Approximately 25% of cytochrome b(5) diffused with a diffusion coefficient of approximately 1 x 10(-8) cm(2)/s, and 50-60% diffused with a diffusion coefficient of approximately 2 x 10(-10) cm(2)/s. Similarly, one-third of annexin V diffused with a diffusion coefficient of approximately 3 x 10(-9) cm(2)/s, and two-thirds diffused with a diffusion coefficient of approximately 4 x 10(-10) cm(2)/s. A model for the interaction of these proteins with the underlying polymer is discussed.     

Lateral diffusion of small compounds in human stratum corneum and model lipid bilayer systems.

An image-based technique of fluorescence recovery after photobleaching (video-FRAP) was used to measure the lateral diffusion coefficients of a series of nine fluorescent probes in two model lipid bilayer systems, dimyristoylphosphatidylcholine (DMPC) and DMPC/cholesterol (40 mol%), as well as in human stratum corneum-extracted lipids. The probes were all lipophilic, varied in molecular weight from 223 to 854 Da, and were chosen to characterize the lateral diffusion of small compounds in these bilayer systems. A clear molecular weight dependence of the lateral diffusion coefficients in DMPC bilayers was observed. Values ranged from 6.72 x 10(-8) to 16.2 x 10(-8) cm2/s, with the smaller probes diffusing faster than the larger ones. Measurements in DMPC/cholesterol bilayers, which represent the most thorough characterization of small-solute diffusion in this system, exhibited a similar molecular weight dependence, although the diffusion coefficients were lower, ranging from 1.62 x 10(-8) to 5.60 x 10(-8) cm2/s. Lateral diffusion measurements in stratum corneum-extracted lipids, which represent a novel examination of diffusion in this unique lipid system, also exhibited a molecular weight dependence, with values ranging from 0.306 x 10(-8) to 2.34 x 10(-8) cm2/s. Literature data showed that these strong molecular weight dependencies extend to even smaller compounds than those examined in this study. A two-parameter empirical expression is presented that describes the lateral diffusion coefficient in terms of the solute's molecular weight and captures the size dependence over the range examined. This study illustrates the degree to which small-molecule lateral diffusion in stratum corneum-extracted lipids can be represented by diffusion in DMPC and DMPC/cholesterol bilayer systems, and may lead to a better understanding of small-solute transport across human stratum corneum.     

Inhomogeneous translational diffusion of monoclonal antibodies on phospholipid Langmuir-Blodgett films.

The translational mobility of fluorescent-labeled monoclonal antibodies specifically bound to supported phospholipid bilayers containing hapten-conjugated phospholipids has been measured as a function of the surface concentration of bound antibodies using fluorescence recovery after photobleaching. Fluorescence recovery curves are fit well by a model that assumes the presence of two populations of antibodies with different lateral diffusion coefficients. The larger diffusion coefficient equals 3.5 x 10(-9) cm2/s, the smaller diffusion coefficient ranges from 1.5 x 10(-9) cm2/s to 2.5 x 10(-10) cm2/s, and the fractional fluorescence recovery associated with the smaller coefficient increases from approximately 0 to approximately 0.7 with increasing concentration of bound antibody. These results suggest that complexes of haptenated phospholipids and antibodies in phospholipid Langmuir-Blodgett films form clusters or domains in a concentration-dependent fashion.     

Lateral diffusion and phase separation in two-dimensional solutions of polymerized butadiene lipid in dimyristoylphosphatidylcholine bilayers. A photobleaching and freeze fracture study.

Mixed vesicles of dimyristoylphosphatidylcholine (DMPC) and a polymerizable lipid containing one diene group per chain are studied by freeze fracture electron microscopy and by the photobleaching (fluorescence recovery after photobleaching) technique. Large thin-walled vesicles of some micron in diameter become more stable after photochemical polymerization. Before polymerization bilayers of the diene lipid exhibit a liquid crystal-to-gel transition at Tg = 31 degrees C. Upon polymerization the transition remains but shifts to a slightly higher temperature (Tg* = 34 degrees C). The transitions in both cases are accompanied by a freezing in of the lateral mobilities. The mixed vesicle exhibits lateral phase separation after polymerization. Before polymerization the two lipids appear miscible at all compositions in the fluid state and at DMPC concentrations at or below 50 mol % in the solid state. After polymerization a two-dimensional solution of the polymer in DMPC is obtained at T greater than Tg*, while lateral phase segregation into DMPC-rich domains and patches of the polymer is observed at T less than Tg*. The domain structure appears identical irrespective of whether polymerization is performed at T greater than Tg or at T less than Tg. A typical value of the diameter of the polymerized lipid domains (approximately 400 A) indicates a rather small aggregation number (N less than 100 monomers). The lateral diffusion coefficient in butadiene-lipid bilayers only decreases from D1 = 3.10(-7) cm2/s to D1 = 8.10(-8) cm2/s (that is by a factor of 4) upon polymerization. This is consistent with the freeze fracture finding of a small aggregation number.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lateral mobility of integral proteins in red blood cell tethers.

The red blood cell membrane is a complex material that exhibits both solid- and liquidlike behavior. It is distinguished from a simple lipid bilayer capsule by its mechanical properties, particularly its shear viscoelastic behavior and by the long-range mobility of integral proteins on the membrane surface. Subject to sufficiently large extension, the membrane loses its shear rigidity and flows as a two-dimensional fluid. These experiments examine the change in integral protein mobility that accompanies the mechanical phenomenon of extensional failure and liquidlike flow. A flow channel apparatus is used to create red cell tethers, hollow cylinders of greatly deformed membrane, up to 36-microns long. The diffusion of proteins within the surface of the membrane is measured by the technique of fluorescence redistribution after photobleaching (FRAP). Integral membrane proteins are labeled directly with a fluorescein dye (DTAF). Mobility in normal membrane is measured by photobleaching half of the cell and measuring the rate of fluorescence recovery. Protein mobility in tether membrane is calculated from the fluorescence recovery rate after the entire tether has been bleached. Fluorescence recovery rates for normal membrane indicate that more than half the labeled proteins are mobile with a diffusion coefficient of approximately 4 x 10(-11) cm2/s, in agreement with results from other studies. The diffusion coefficient for proteins in tether membrane is greater than 1.5 x 10(-9) cm2/s. This dramatic increase in diffusion coefficient indicates that extensional failure involves the uncoupling of the lipid bilayer from the membrane skeleton.     

Fast diffusion along defects and corrugations in phospholipid P beta, liquid crystals.

The diffusion of a fluorescent lipid analogue in liquid crystals of the anisotropic P beta, phase of dimyristoylphosphatidylcholine (DMPC) had been found to be highly variable, suggesting structural defect pathways. Fluorescence photobleaching recovery (FPR) experiments imply two effective diffusion pathways with coefficients differing by at least 100. This is consistent with fast diffusion along submicroscopic bands of disordered material ("defects") in the bilayer corrugations characteristic of this phase. Due to strains during transformation from the L alpha phase, the axis of the corrugations is ordinarily disrupted by mosaic patches rotationally disoriented within the mean plane of the molecular bilayers, although larger oriented domains are sometimes adventitiously aligned into microscopically visible striped textures. The corrugations are also systematically aligned along positive disclinations pairs or "oily streaks." Thus, fast diffusion occurs parallel to the disclination lines and along the textured stripes. FPR results yield an upper limit on the effective diffusion in the ordered material of D less than or equal to 2 X 10(-16) cm2/s at 22 degrees C, D less than or equal to 3 X 10(-17) cm2/s at 13 degrees C. In contrast the diffusion coefficient along defect pathways where disordered ribbons are aligned is D approximately 4 X 10(-11) cm2/s at 16 degrees C.     

Membrane insertion and lateral diffusion of fluorescence-labelled cytochrome c oxidase subunit IV signal peptide in charged and uncharged phospholipid bilayers.

The synthetic 25-residue signal peptide of cytochrome c oxidase subunit IV was labelled with the fluorophor 7-nitrobenz-2-oxa-1,3-diazole (NBD) at its single cysteine residue. Addition of small unilamellar vesicles of 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC) to the labelled peptide resulted in a shift of the NBD excitation and emission spectra to shorter wavelengths. Binding of the peptide to the vesicles was measured by the increase in the fluorescence emission yield. A surface partition constant of (3.9 +/- 0.5) x 10(3) M-1 was derived from these titrations. When the membrane contained, in addition to POPC, negatively charged 1-palmitoyl 2-oleoyl phosphatidylglycerol (POPG), the NBD fluorescence spectra were further shifted to shorter wavelengths and exhibited increased quantum yields. The apparent partition constants were increased to 10(4)-10(5) M-1 for vesicles with 20 or 100 mol% POPG. Lateral diffusion of the peptide was measured by fluorescence recovery after photobleaching in multibilayers of POPC, POPG, POPC/POPG (4:1) and 1,2-dimyristoyl phosphatidylcholine. The lateral diffusion coefficients of the peptide in bilayers of POPC (8 x 10(-8) cm2/s at 21 degrees C) were 1.5-1.6-fold greater than those of NBD-labelled phospholipids (5 x 10(-8) cm2/s at 21 degrees C), but 1.5-1.8-fold smaller (3 x 10(-8) cm2/s in 20% POPG and at 21 degrees C) than the lipid diffusion coefficients in the negatively charged bilayers. It is concluded that the signal peptide associates with phospholipid bilayers in two different forms, which depend on the lipid charge. The experiments with POPC bilayers are well explained by a model in which the peptide partitions into the region of the phospholipid head-groups and diffuses along the membrane/water interface. If POPG is present in the membrane, electrostatic attractions between the basic residues of the peptide and the acidic lipid head-groups result in a deeper penetration of the bilayer. For this case, two models that are both consistent with the experimental data are discussed, in which the peptide either forms an oligomer of three to six partially helical membrane-spanning monomers, or inserts into the bilayer with its amphiphilic helical segment aligned parallel to the plane of the membrane and located near the head-group and outer hydrocarbon region of the bilayer.     

Fluid-phase connectivity and translational diffusion in a eutectic, two-component, two-phase phosphatidylcholine bilayer

In recent work [Vaz, W.L.C., Melo, E.C.C., & Thompson, T.E. (1989) Biophys. J. 56, 869-876] we have shown that translational diffusion studies using fluorescence recovery after photobleaching (FRAP) provide information concerning domain structures and fluid-phase connectivity in lipid bilayers in which solid and fluid phases coexist. In the present paper, translational diffusion of the fluid-phase-soluble, solid-phase-insoluble fluorescent lipid derivative N-(7-nitrobenzoxa-2,3-diazol-4-yl) dilauroyl-phosphatidylethanolamine and the fluid-phase connectivity are examined in lipid bilayers prepared from binary mixtures of 1-docosanoyl-2-dodecanoylphosphatidylcholine (C22:0C12:0PC) and 1,2-diheptadecanoylphosphatidylcholine (di-C17:0PC) by using FRAP. The phosphatidylcholine mixture used provides a eutectic system with a eutectic point at a composition of about 0.4 mole fraction of di-C17:0PC and a temperature of about 37 degrees C [Sisk, R.B., Wang, Z.Q., Lin, H.N., & Huang, C.H. (1990) Biophys. J. 58, 777-783]. Two regions in temperature and composition, respectively below and above 0.4 mole fraction of di-C17:0PC, where fluid and solid phases coexist in the same lipid bilayer, are available for examination of fluid-phase connectivity. In mixtures containing less than 0.4 mole fraction of di-C17:0PC the fluid phase coexists with a mixed interdigitated Lc gel phase composed mostly of C22:0C12:0PC, whereas in mixtures containing greater than 0.4 mole fraction of di-C17:0PC the fluid phase coexists with a P beta' gel phase mostly composed of di-C17:0PC. When the solid phase is a P beta' gel phase, the temperature of fluid-phase connectivity for the mixtures lies close to the fluidus, which means that a small (approximately 20%) mass fraction of solid phase can divide the large bulk of the bilayer that is fluid into nonconnected domains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diffusion of dihydropyridine calcium channel antagonists in cardiac sarcolemmal lipid multibilayers.

A membrane bilayer pathway model has been proposed for the interaction of dihydropyridine (DHP) calcium channel antagonists with receptors in cardiac sarcolemma (Rhodes, D.G., J.G. Sarmiento, and L.G. Herbette. 1985. Mol. Pharmacol. 27:612-623) involving drug partition into the bilayer with subsequent receptor binding mediated (though probably not rate-limited) by diffusion within the bilayer. Recently, we have characterized the partition step, demonstrating that DHPs reside, on a time-average basis, near the bilayer hydrocarbon core/water interface. Drug distribution about this interface may define a plane of local concentration for lateral diffusion within the membrane. The studies presented herein examine the diffusional dynamics of an active rhodamine-labeled DHP and a fluorescent phospholipid analogue (DiIC16) in pure cardiac sarcolemmal lipid multibilayer preparations as a function of bilayer hydration. At maximal bilayer hydration, the drug diffuses over macroscopic distances within the bilayer at a rate identical to that of DiI (D = 3.8 X 10(-8) cm2/s), demonstrating the overall feasibility of the membrane diffusion model. The diffusion coefficients for both drug and lipid decreased substantially as the bilayers were dehydrated. While identical at maximal hydration, drug diffusion was significantly slower than that of DiIC16 in partially dehydrated bilayers, probably reflecting differences in mass distribution of these probes in the bilayer.     

Repulsive interactions between uncharged bilayers. Hydration and fluctuation pressures for monoglycerides.

Pressure versus distance relations have been obtained for solid (gel) and neat (liquid-crystalline) phase uncharged lipid bilayers by the use of x-ray diffraction analysis of osmotically stressed monoglyceride aqueous dispersions and multilayers. For solid phase monoelaidin bilayers, the interbilayer repulsive pressure decays exponentially from a bilayer separation of approximately 7 A at an applied pressure of 3 x 10(7) dyn/cm2 to a separation of approximately 11 A at zero applied pressure, where an excess water phase forms. The decay length is approximately 1.3 A, which is similar to the value previously measured for gel phase phosphatidylcholine bilayers. This implies that the decay length of the hydration pressure does not depend critically on the presence of zwitterionic head groups in the bilayer surface. For liquid-crystalline monocaprylin, the repulsive pressure versus distance curve has two distinct regions. In the first region, for bilayer separations of approximately 3-8 A and applied pressures of 3 x 10(8) to 4 x 10(6) dyn/cm2, the pressure decays exponentially with a decay length of approximately 1.3 A. In the second region, for bilayer separations of approximately 8-22 A and applied pressures of 4 x 10(6) to 1 x 10(5) dyn/cm2, the pressure decays much more gradually and is inversely proportional to the cube of the distance between bilayers. These data imply that two repulsive pressures operate between liquid-crystalline monocaprylin bilayers, the hydration pressure, which dominates at small (3-8 A) bilayer separations, and the fluctuation pressure, which dominates at larger bilayer separations (greater than 8 A) and strongly influences the hydration properties of the liquid-crystalline bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Size dependence of the translational diffusion of large integral membrane proteins in liquid-crystalline phase lipid bilayers. A study using fluorescence recovery after photobleaching

The translational diffusion of bovine rhodopsin, the Ca2+-activated adenosinetriphosphatase of rabbit muscle sarcoplasmic reticulum, and the acetylcholine receptor monomer of Torpedo marmorata has been examined at a high dilution (molar ratios of lipid/protein greater than or equal to 3000/1) in liquid-crystalline phase phospholipid bilayer membranes by using the fluorescence recovery after photobleaching technique. These integral membrane proteins having molecular weights of about 37 000 for rhodopsin, about 100 000 for the adenosinetriphosphatase, and about 250 000 for the acetylcholine receptor were reconstituted into membranes of dimyristoylphosphatidylcholine (rhodopsin and acetylcholine receptor), soybean lipids (acetylcholine receptor), and a total lipid extract of rabbit muscle sarcoplasmic reticulum (adenosinetriphosphatase). The translational diffusion coefficients of all the proteins at 310 K were found to be in the range (1-3) X 10(-8) cm2/s. In consideration of the sizes of the membrane-bound portions of these proteins, this result is in agreement with the weak dependence of the translational diffusion coefficient upon diffusing particle size predicted by continuum fluid hydrodynamic models for the diffusion in membranes [Saffman, P. G., & Delbrück, M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3111-3113]. Lipid diffusion was also examined in th same lipid bilayers with the fluorescent lipid derivative N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dimyristoylphosphatidylethanolamine. The translational diffusion coefficient for this lipid derivative was found to be in the range (9-14) X 10(-8) cm2/s at 310 K. In consideration of the dimensions of the lipid molecule, this value for the lipid diffusion coefficient is in agreement with the continuum fluid hydrodynamic model only if a near-complete slip boundary condition is assumed at the bilayer midplane. Alternatively, kinetic diffusion models [Tr?uble, H., & Sackmann. E. (1972) J. Am. Chem. Soc. 94, 4499-4510] may have to be invoked to explain the lipid diffusion behavior.     

Incorporation of fluorescent gangliosides into human fibroblasts: mobility, fate, and interaction with fibronectin

Rhodamine- and fluorescein-labeled gangliosides were used as probes to investigate the distribution, dynamics, and fate of plasma membrane-bound gangliosides on cultured human fibroblasts. When sparse cultures of fibroblasts were incubated with the fluorescent ganglioside derivatives, their surfaces became highly fluorescent. The fluorescent gangliosides were taken up by the cells in a time- and temperature-dependent manner and were not removed from the cell surface by trypsin or serum. Thus, the gangliosides appeared to be stably incorporated into the lipid bilayer of the plasma membrane. Fluorescent photobleaching recovery measurements showed that the inserted gangliosides were free to diffuse in the plane of the membrane with a high diffusion coefficient of approximately 10(-8) cm2/s. When the ganglioside-treated cells were washed and incubated in fresh medium, the surface gangliosides became internalized with time, and localized in the perinuclear region of the fibroblasts. In dense cultures of fibroblasts, a large fraction of the fluorescent gangliosides were organized in a fibrillar network and were immobile on the time scale of fluorescent photobleaching recovery measurements. Using antifibronectin antibodies and indirect immunofluorescence, these gangliosides were found to co-distribute with fibrillar fibronectin. Thus, exogenous gangliosides appear to be stably inserted into the lipid bilayer of the plasma membrane and to diffuse freely in its plane as well as form a less mobile state with the fibrillar networks of fibronectin associated with the cells.     

SNARE-driven, 25-millisecond vesicle fusion in vitro

Docking and fusion of single proteoliposomes reconstituted with full-length v-SNAREs (synaptobrevin) into planar lipid bilayers containing binary t-SNAREs (anchored syntaxin associated with SNAP25) was observed in real time by wide-field fluorescence microscopy. This enabled separate measurement of the docking rate k(dock) and the unimolecular fusion rate k(fus). On low t-SNARE-density bilayers at 37 degrees C, docking is efficient: k(dock) = 2.2 x 10(7) M(-1) s(-1), approximately 40% of the estimated diffusion limited rate. Full vesicle fusion is observed as a prompt increase in fluorescence intensity from labeled lipids, immediately followed by outward radial diffusion (D(lipid) = 0.6 microm2 s(-1)); approximately 80% of the docked vesicles fuse promptly as a homogeneous subpopulation with k(fus) = 40 +/- 15 s(-1) (tau(fus) = 25 ms). This is 10(3)-10(4) times faster than previous in vitro fusion assays. Complete lipid mixing occurs in <15 ms. Both the v-SNARE and the t-SNARE are necessary for efficient docking and fast fusion, but Ca2+ is not. Docking and fusion were quantitatively similar on syntaxin-only bilayers lacking SNAP25. At present, in vitro fusion driven by SNARE complexes alone remains approximately 40 times slower than the fastest, submillisecond presynaptic vesicle population response.     

Translocation of phospholipids and dithionite permeability in liquid-ordered and liquid-disordered membranes

We present a detailed study of the translocation rate of two headgroup-labeled phospholipid derivatives, one with two acyl chains, NBD-DMPE, and the other with a single acyl chain, NBD-lysoMPE, in lipid bilayer membranes in the liquid-disordered state (POPC) and in the liquid-ordered states (POPC/cholesterol (Chol), molar ratio 1:1, and sphingomyelin (SpM)/Chol, molar ratio 6:4). The study was performed as a function of temperature and the thermodynamic parameters of the translocation process have been obtained. The most important findings are 1), the translocation of NBD-DMPE is significantly faster than the translocation of NBD-lysoMPE for all bilayer compositions and temperatures tested; and 2), for both phospholipid derivatives, the translocation in POPC bilayers is approximately 1 order of magnitude faster than in POPC/Chol (1:1) bilayers and approximately 2-3 orders of magnitude faster than in SpM/Chol (6:4) bilayers. The permeability of the lipid bilayers to dithionite has also been measured. In liquid disordered membranes, the permeability rate constant obtained is comparable to the translocation rate constant of NBD-DMPE. However, in liquid-ordered bilayers, the permeability of dithionite is significantly faster then the translocation of NBD-DMPE. The change in enthalpy and entropy associated with the formation of the activated state in the translocation and permeation processes has also been obtained.     

Obstructed diffusion in phase-separated supported lipid bilayers: a combined atomic force microscopy and fluorescence recovery after photobleaching approach

Proteins and other macromolecules are believed to hinder molecular lateral diffusion in cellular membranes. We have constructed a well-characterized model system to better understand how obstacles in lipid bilayers obstruct diffusion. Fluorescence recovery after photobleaching was used to measure the lateral diffusion coefficient in single supported bilayers composed of mixtures of 1,2-dilauroylphosphotidylcholine (DLPC) and 1,2-distearoylphosphotidylcholine (DSPC). Because these lipids are immiscible and phase separate at room temperature, a novel quenching technique allowed us to construct fluid DLPC bilayers containing small disk-shaped gel-phase DSPC domains that acted as obstacles to lateral diffusion. Our experimental setup enabled us to analyze the same samples with atomic force microscopy and exactly characterize the size, shape, and number of gel-phase domains before measuring the obstacle-dependent diffusion coefficient. Lateral obstructed diffusion was found to be dependent on obstacle area fraction, size, and geometry. Analysis of our results using a free area diffusion model shows the possibility of unexpected long-range ordering of fluid-phase lipids around the gel-phase obstacles. This lipid ordering has implications for lipid-mediated protein interactions in cellular membranes.     

A photobleaching recovery study of glucocorticoid effects on lateral mobilities of a lipid analog in S3G HeLa cell membranes

Treatment of the S3G strain of HeLa cells with dexamethasone inhibits cholesterol synthesis and thus results in decreased plasma membrane cholesterol-to-protein ratios. Incubation of HeLa cells with dexamethasone for 24 h lowers the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) in intact cell plasma membranes and isolated plasma membranes (Johnston, D. and Melnykovych, G. (1980) Biochim. Biophys. Acta 596, 320–324). We have examined the effect of dexamethasone treatment of S3G HeLa cells on the lateral diffusion of the fluorescent lipid analogue 3,3′-dioctadecylindocarbocyanine iodide (DiI) by the fluorescence photobleaching recovery technique. The lateral diffusion of DiI was measured in cells 0, 2, 6, 12, and 24 h following treatment with dexamethasone and in cells identically handled without dexamethasone at 37°C. The diffusion constants of DiI in the treated and untreated cell membranes at zero time were (4.52±0.30) · 10^?9 cm²/s and (4.56±0.24) · 10^?9 cm²/s, respectively. There was no significant change in the lateral diffusion of DiI in the untreated cells over the 24 h period. The lateral diffusion of the lipid probe in the dexamethasone-treated cells began to increase 6 h following treatment and reached (6.43±0.27) · 10^?9 cm²/s at 24 h. The lateral diffusion of DiI was also measured at 25, 17, 10 and 4°C following 24 h incubation with and without dexamethasone. The effect of dexamethasone treatment on the lipid probe lateral diffusion observed at 37°C is decreased at 25°C and reversed in direction at 10 and 4°C. These results agree with those obtained in artificial systems containing varying amounts of cholesterol and support the suggestion that cholesterol acts to suppress phospholipid phase changes in animal cells. The lateral diffusion of DiI localized as a monolayer at a mineral oil-water interface was measured by fluorescence photobleaching recovery. The resulting data and the viscosity of the mineral oil were used to calculate the microviscosities of the plasma membranes of untreated and dexamethasone-treated cells at 25°C. Membrane microviscosities were also calculated from the fluorescence polarization studies cited above. In both cases the dexamethasone treatment reduced the apparent microviscosity by approximately 25%. However, the absolute microviscosity values obtained by the two techniques differ by a factor of 3.     

Trafficking of endothelial nitric-oxide synthase in living cells. Quantitative evidence supporting the role of palmitoylation as a kinetic trapping mechanism limiting membrane diffusion.

To examine endothelial nitric-oxide synthase (eNOS) trafficking in living endothelial cells, the eNOS-deficient endothelial cell line ECV304 was stably transfected with an eNOS-green fluorescent protein (GFP) fusion construct and characterized by functional, biochemical, and microscopic analysis. eNOS-GFP was colocalized with Golgi and plasma membrane markers and produced NO in response to agonist challenge. Localization in the plasma membrane was dependent on the palmitoylation state, since the palmitoylation mutant of eNOS (C15S/C26S eNOS-GFP) was excluded from the plasma membrane and was concentrated in a diffuse perinuclear pattern. Fluorescence recovery after photobleaching (FRAP) revealed eNOS-GFP in the perinuclear region moving 3 times faster than the plasmalemmal pool, suggesting that protein-lipid or protein-protein interactions are different in these two cellular domains. FRAP of the palmitoylation mutant was two times faster than that of wild-type eNOS-GFP, indicating that palmitoylation was influencing the rate of trafficking. Interestingly, FRAP of C15S/C26S eNOS-GFP but not wild-type eNOS-GFP fit a model of protein diffusion in a lipid bilayer. These data suggest that the regulation of eNOS trafficking within the plasma membrane and Golgi are probably different mechanisms and not due to simple diffusion of the protein in a lipid bilayer.     

Lipid diffusibility in the intact erythrocyte membrane

The lateral diffusion of fluorescent lipid analogues in the plasma membrane of intact erythrocytes from man, mouse, rabbit, and frog has been measured by fluorescence photobleaching recovery (FPR). Intact cells from dystrophic, normoblastic, hemolytic, and spherocytotic mouse mutants; from hypercholesterolemic rabbits and humans; and from prenatal, neonatal, and juvenile mice have been compared with corresponding normals. The lateral diffusion coefficient (D) for 3,3'-dioctadecylindodicarbocyanine iodide (DiI[5]) in intact normal human erythrocytes is D = 8.2 +/- 1.2 X 10(-9) cm2/s at 25 degrees C and D = 2.1 +/- 0.4 X 10(-8) cm2/s at 37 degrees C, and varies approximately 50-fold between 1 degree and 42 degrees C. The diffusion constants of lipid analogue rhodamine-B phosphatidylethanolamine (RBPE) are about twice those of DiI[5]. The temperature dependence and magnitude of D vary by up to a factor of 3 between species and are only influenced by donor age in prenatals. DiI[5] diffusibility is not perturbed by the presence of calcium or local anesthetics or by spectrin depletion (via mutation). However, lipid-analogue diffusibility in erythrocyte ghosts may differ from intact cells. Dietary hypercholesterolemia in rabbits reduces the diffusion coefficient and eliminates the characteristic break in Arrhenius plots of D found in all other cells studied except frog.