Particulate enhanced membrane uptake of 1,2-benzanthracene observed by fluorescence spectroscopy: a possible role in co-carcinogenesis.

In recognition of the co-carcinogenic effects of particulate matter and chemical carcinogens, we investigated the effect of particulate silica on the rates of membrane uptake of 1,2-benzanthracene. The fluorescence emission spectra and the apparent quantum yields of benzanthracene and dependent upon adsorption to silica and upon the surface density of benzanthracene on the silica. The fluorescence spectral shifts which occur upon transfer of benzanthracene from the silica surface to phospholipid vesicles provided a convenient means to quantitate the membrane uptake of benzanthracene from particulates. The rate of benzanthracene uptake by dipalmitoyl-L-alpha-phosphatidylcholine vesicles was independent of the concentration of lipid, indicating that the rate-limiting step may involve its solubilization in the aqueous phase. These uptake rates were also independent of the surface density of benzanthracene on the silica, indicating that the benzathracene molecules are dispersed uniformly on the silica surface. Rates of membrane uptake of benzanthracene from the crystalline, microcrystalline, and the silica-absorbed states were compared, and are greatly enhanced by a reduction in crystal size. Silica-adsorbed benzanthracene had the most rapid rate of membrane uptake. Silica did not cause disruption of the lipid vesicles. These results indicate that particulates can enhance the cellular availability of the chemical carcinogens.     

Benzo[a]pyrene uptake into rat liver microsomes: Effects of adsorption of benzo[a]pyrene to asbestos and non-fibrous mineral particulates

The fluorescence yield of benzo[a]pyrene (BP) increases dramatically upon its transfer from the surface of particulates to rat liver microsomes. Adsorption of BP to Canadian chrysotile, anthophyllite, hematite and silica results in greatly enhanced uptake rates into microsomes when compared to uptake from a microcrystalline dispersion of BP. The fibrous minerals chrysotile and anthophyllite were more effective than silica and hematite in enhancing BP uptake. Simple mixtures of BP microcrystals and particles did not display enhanced transport, indicating that adsorption of BP to the particulate surface is necessary for enhanced microsomal uptake. BP was not released into microsomes from carbon black.We suggest that particulate-enhanced availability of BP may be of significance in the co-carcinogenesis between particulates and polynuclear aromatic hydrocarbons. However, other mechanisms are also possible, and are not excluded by our experiments. The fluorescence methodology described in this paper provides a novel and convenient means to quantify microsomal uptake of BP and thereby investigate further the mechanisms of cocarcinogenesis.     

A fluorescence spectroscopy study on the interactions of the TAT-PTD peptide with model lipid membranes

Protein-transduction domains (PTDs) have been shown to translocate into and through the living cells in a rapid manner by an as yet unknown mechanism. Regardless of the mechanism of translocation, the first necessary step must be binding of the PTD peptide to the surface of the lipid membrane. We used fluorescence spectroscopy to study the interaction between PTD of the HIV-1 Tat protein (TAT-PTD; residues 47-60 of Tat, fluorescently labeled with tryptophan) and the lipid bilayer labeled with various fluorescence membrane probes. The TAT-PTD tryptophan exhibited a decrease in fluorescence intensity and an increase in anisotropy upon interaction with lipid bilayers. The fluorescence changes were linearly proportional to the density of negative charge in the membrane. Kinetic analysis of the interaction showed two apparent dissociation constants. The value of one dissociation constant (Kd1 = 2.6 +/- 0.6 microM), which accounted for 24% of the interaction, was found to be independent of the negative charge density, suggesting its nonelectrostatic nature. The value of the second dissociation constant (Kd2), which accounted for 76% of the interaction, decreased linearly from 610 +/- 150 to 130 +/- 30 microM with an increase in negative charge density from 0 to 25 mol %, suggesting this interaction is electrostatic in nature. Even though the binding was predominantly electrostatic, it could not be reversed by high salt, indicating the presence of a second, irreversible, step in the interaction with lipid. When TAT-PTD was bound to lipid vesicles labeled with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), fluorescence resonance energy transfer between the tryptophan and the probe occurred at a distance of 3.4 nm. No change in fluorescence anisotropy of either TMA-DPH or DPH was observed upon the interaction with TAT-PTD, indicating no significant disruption or perturbation of the lipid bilayer by the peptide. TAT-PTD did not cause dissipation of membrane potential (165 mV, negative inside). Inclusion of 3% pyrene-labeled phosphatidylglycerol (pyrene-PG) in the membrane revealed that TAT-PTD preferentially bound to the membrane in the liquid state. We conclude that membrane fluidity is an important physicochemical parameter, which may regulate binding of TAT-PTD to the membrane.     

Role of oxysterol structure on the microdomain-induced microsolubilization of phospholipid membranes by apolipoprotein A-I

Oxygenated derivatives of cholesterol, oxysterols, have different physicochemical properties and three-dimensional shapes. The kinetics of microsolubilization of dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles by apolipoprotein A-I (apoA-I) to form discoidal high-density lipoproteins (rHDL) was dramatically affected by oxysterol chemical structure. Under the experimental conditions of varying oxysterol chemical structure, sterol concentration, and the lipid phase state of DMPC, the kinetics varied over 3 orders of magnitude. Some oxysterols behaved similarly to cholesterol and increased the rate of microsolubilization; however, they were not as effective as cholesterol. Other oxysterols greatly inhibited this process. In general, there was no correlation of the rates with membrane fluidity as measured by fluorescence polarization. The rate of DMPC microsolubilization by apoA-I is highly dependent upon the presence of lattice defects in the membrane surface that occur due to imperfect packing of coexisting lipid phases. The differential ability of various oxysterols to induce the formation of an ordered lipid phase is the probable basis for their effects on the rates of DMPC microsolubilization. There was no effect of oxysterol chemical structure on the structure of the equilibrium rHDL products; however, there was a dramatic effect of sterol concentration on rHDL particle size. Different oxysterols regulate the kinetics of apoA-I membrane association by altering structural microheterogeneity at the membrane surface. However, once the kinetic barrier is overcome, the particle sizes of rHDL products formed are determined solely by the amount of sterol presence.     

Conformation and self-assembly of a nystatin nitrobenzoxadiazole derivative in lipid membranes

Nystatin is a polyene (tetraene) macrolide antibiotic presenting antifungal activity that acts at the cellular membrane level. In the present study, we report the interaction of this antibiotic labelled at its amine group with 7-nitrobenz-2-oxa-1,3-diazole (NBD-Nys) with sterol-free and ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) large unilamellar vesicles (LUV). The mean tetraene to NBD separating distance determined from fluorescence energy transfer measurements increased from 18 to 25.6 A upon antibiotic binding to the lipid vesicles, indicating that the monomeric labelled antibiotic adopts a more extended conformation in its lipid-bound state than in aqueous solution. The oligomeric state of membrane-bound NBD-Nys was also studied by resonance energy homotransfer between the NBD fluorophores. The decrease measured in its steady state fluorescence anisotropy upon increasing the surface concentration of the NBD-Nys is shown to be consistent with a random distribution of molecules on the surface of the liposomes. This data contradicts the sharp increase measured for nystatin mean fluorescence lifetime in the presence of 10 mol% ergosterol-containing POPC LUV within the same antibiotic concentration range and which is known to report nystatin oligomerization in the lipid vesicles. Therefore, we conclude that the amine group of nystatin is an essential requisite for the supramolecular organization/pore formation of this antibiotic.     

Insertion of fluorescent phosphatidylserine into the plasma membrane of red blood cells. Recognition by autologous macrophages

The interaction of macrophages with red blood cells (RBC) displaying phosphatidylserine (PS) in their surface membranes was investigated after the transfer of an exogenously supplied fluorescent lipid analog to the RBC. Nonfluorescent (quenched) lipid vesicles were formed by ultrasonication from 1-acyl-2-[(N-4-nitro-benzo-2-oxa-1,3 diazole)aminocaproyl]phosphatidyl-serine (NBD-PS) or 1-acyl-2[(N-4-nitrobenzo-2-oxa-1,3 diazole)aminocaproyl]phosphatidylcholine (NBD-PC). The interaction of these vesicles with RBC was monitored as a function of vesicle concentration by assessment of the degree to which cell-associated lipid fluorescence was dequenched after vesicle treatment. When vesicle concentrations of less than 100 ng/ml were used, lipid fluorescence was largely dequenched, indicating that lipid transfer was the predominant mechanism of both NBD-PS and NBD-PC uptake; however, when vesicle concentrations were increased to greater than 100 ng/ml, a concentration-dependent increase in the fraction of quenched cell-associated lipid was observed, indicating that another mechanism, possibly vesicle-cell adhesion, also occurred. Using NBD-PS at concentrations at which dilution of all the phospholipid analog in the recipient cell membrane could be unequivocally confirmed, we observed that the uptake of NBD-PS-treated RBC by macrophages was increased 5-fold over that of controls, whereas the uptake of RBC containing an equivalent amount of exogenously supplied NBD-PC was unaltered. Furthermore, preincubation of macrophage monolayers with vesicles containing PS resulted in a approximately 60% inhibition in the uptake of NBD-PS-treated RBC, whereas no inhibition in the uptake of control, opsonized, or NBD-PC-treated RBC was observed. These findings suggest that PS in the outer leaflet of RBC might serve as a signal for triggering their recognition by macrophages.     

The role of the transmembrane domain of silicanin-1: Reconstitution of the full-length protein in artificial membranes

Biosilica formation in diatoms is a membrane-confined process that occurs in so-called silica deposition vesicles (SDVs). As SDVs have as yet not been successfully isolated, the impact of the SDV membrane on silica morphogenesis is not well understood. However, recently the first SDV transmembrane protein, silicanin-1 (Sin1) has been identified that appears to be involved in biosilica formation. In this study, we recombinantly expressed and isolated full-length Sin1 from E. coli and investigated its reconstitution behavior in artificial membranes. A reconstitution efficiency in vesicles of up to 80% was achieved by a co-micellization method. By using a chymotrypsin digest, the orientation of Sin1 in unilamellar vesicles was analyzed indicating a positioning of the large N-terminal domain to the outside of the vesicles. These proteoliposomes were capable of precipitating silica in the presence of long-chain polyamines. Supported lipid bilayers were produced by proteoliposome spreading on lipid monolayers to form continuous lipid bilayers with Sin1 confined to the membrane. Successful Sin1 reconstitution into these planar membranes was shown by means of immunostaining with purified primary anti-Sin1 and secondary fluorescent antibodies. The established planar model membrane system, amenable for surface sensitive and microscopy techniques, will pave the way to investigate SDV-membrane interactions with other SDV associated biomolecules and its role in silica biogenesis.     

In vitro interaction of Zajdela ascites hepatoma cells with lipid vesicles.

We studied the in vitro interaction between Zajdela ascites hepatoma cells and small unilamellar vesicles, consisting of 14C-labeled phosphatidylacholine, cholesterol, and phosphatidylserine (molar ratio 5 : 4 : 1), containing high intravesicular concentrations of carboxyfluorescein or fluorescein isothiocyanate tagged dextran. The entrapped markers were found to be associated with the cells to a lesser degree than the vesicle membrane marker. This discrepancy, which is slightly less pronounced for fluorescein isothiocyanate tagged dextran than for carboxyfluorescein, increases with incubation time and decreases with increasing vesicle lipid concentration in the incubation mixture. Vesicle-plasma membrane exchange of the vesicle lipid marker could not entirely explain the observed discrepancy. It is tentatively concluded that the gap mainly arises from a selective loss of entrapped dyes from vesicles actually interacting with the cell surface. Both spectrofluorimetry and fluorescence microscopic observations, as well as the relative insensitivity of vesicle uptake towards the presence of metabolic inhibitors, exclude a major contribution of endocytosis as a vesicle uptake route. We therefore conclude that vesicles are primarily internalized by a vesicle-cell fusion-like process. The observed discrepancy in uptake between entrapped materials and vesicle lipid is discussed in terms of a two-site vesicle-cell surface interaction model.     

The use of cobalt ions as a collisional quencher to probe surface charge and stability of fluorescently labeled bilayer vesicles

Co2+ quenched the fluorescence of the lipid probes NBD-phosphatidylethanolamine (NBD-PE) and lissamine-rhodamine phosphatidylethanolamine (N-Rh-PE) incorporated into lipid vesicles, according to a collisional quenching mechanism in agreement with the Stern-Vollmer law. The quenching coefficient (Q) for NBD-PE, incorporated into uncharged phosphatidylcholine (PC) vesicles was 13.8 M-1. This value was equal to the quenching coefficient of water-soluble NBD-taurine in aqueous solution, indicating that Co2+ was readily accessible to the outer surface of PC vesicles. In phosphatidylserine-phosphatidylethanolamine (PS-PE) (1:1) vesicles, quenching was also proportional to Co2+ concentration but Q was 114 mM-1, some 8000-fold smaller. Using the Gouy-Chapman-Stern model we demonstrated that the surface density of Co2+ bound to lipid was linear with Co2+ concentration in the medium up to 7%. Co2+-associated phospholipid would in turn quench NBD-PE or N-Rh-PE by collisional quenching with lateral diffusion. We investigated the ability of Co2+ to permeate PS-PE (1:1) vesicles. Co2+ quenched fluorophores on the outer surface of large unilamellar vesicles, formed by reverse-phase evaporation. In small unilamellar vesicles Co2+ quenched probes on both outer and inner surfaces, indicating rapid permeation of the ions into the vesicles. Using stopped-flow rapid mixing, we measured the rate of influx of Co2+, and correcting for surface potential using the Gouy-Chapman-Stern model, we calculated a permeability coefficient of 10(-12) cm/s for Co2+ concentrations below 300 microM. Above this concentration, there was a very steep rise in the permeability coefficient, indicating that binding of Co2+ induces defects in the bilayer of these vesicles. This may be related to the ability of the vesicles to undergo membrane fusion. A method for calculating the membrane surface potential from Co2+ quenching data is presented.     

Electrochemical potential and ion transport in vesicles of yeast plasma membrane

Vesicles from yeast plasma membrane were prepared according to Franzusoff and Cirillo [1983) J. Biol. Chem. 258, 3608), with slight modifications. When Mg-ATP was added, this preparation was able to generate a membrane potential, that was sensitive to inhibitors of the yeast H+-ATPase and uncouplers, and could be decreased by the addition of permeant anions, as measured by the fluorescence changes of the dye oxonol V. The addition of ATP could also generate a pH gradient, detectable by the fluorescence changes of the monitor aminochloromethoxyacridine. This gradient was sensitive to inhibitors of ATPase and uncouplers, and could be increased by the addition of permeant anions to the incubation mixture. When the vesicles were loaded with KCl, an increased rate of K+ efflux was produced upon the addition of ATP. Cytochrome oxidase from bovine heart could be reconstituted into the vesicles and was shown to generate a membrane potential difference, negative inside, evidenced by the fluorescence quenching of the cyanide dipropylthiacarbocyanine and the uptake of tetraphenylphosphonium. Besides, in these vesicles, K+ and Rb+, but not Na+ or NH+4 could decrease the quenching of fluorescence and the uptake of tetraphenylphosphonium produced when the electron-donor system was present. In the vesicles in which cytochrome oxidase was incorporated, upon the addition of cytochrome c and ascorbate, the uptake of 86Rb+ could be demonstrated also. This uptake was found to be saturable and inhibited by K+, and to a lesser degree by Na+. The results obtained indicate that these vesicles are reasonably sealed and capable of generating and maintaining a membrane potential. The membrane potential could be used to drive ions across the membrane of the vesicles, indicating the presence and functionality of the monovalent cation carrier. The vesicles, in general terms seem to be suitable for studying transport of ions and metabolites in yeast.     

In vitro interaction of zajdela ascites hepatoma cells with lipid vesicles

We studied the in vitro interaction between Zajdela ascites hepatoma cells and small unilamellar vesicles, consisting of ¹⁴C-labeled phosphatidylcholine, cholesterol, and phosphatidylserine (molar ratio 5: 4: 1), containing high intravesicular concentrations of carboxyfluorescein or fluorescein isothiocyanate tagged dextran.The entrapped markers were found to be associated with the cells to a lesser degree than the vesicle membrane marker. This discrepancy, which is slightly less pronounced for fluorescein isothiocyanate tagged dextran than for carboxyfluorescein, increases with incubation time and decreases with increasing vesicle lipid concentration in the incubation mixture. Vesicle-plasma membrane exchange of the vesicle lipid marker could not entirely explain the observed discrepancy. It is tentatively concluded that the gap mainly arises from a selective loss of entrapped dyes from vesicles actually interacting with the cell surface. Both spectrofluorimetric and fluorescence microscopic observations, as well as the relative insensitivity of vesicle uptake towards the presence of metabolic inhibitors, exclude a major contribution of endocytosis as a vesicle uptake route. We therefore conclude that vesicles are primarily internalized by a vesicle-cell fusion-like process. The observed discrepancy in uptake between entrapped materials and vesicle lipid is discussed in terms of a two-site vesicle cell surface interaction model.     

Interaction of mammalian Hsp22 with lipid membranes

Hsp22/HspB8 is a member of the small heat-shock protein family, whose function is not yet completely understood. Our immunolocalization studies in a human neuroblastoma cell line, SK-N-SH, using confocal microscopy show that a significant fraction of Hsp22 is localized to the plasma membrane. We therefore investigated its interactions with lipid vesicles in vitro. Intrinsic tryptophan fluorescence is quenched in the presence of lipid vesicles derived from either bovine brain lipid extract or purified lipids. Time-resolved fluorescence studies show a decrease in the lifetimes of the tryptophan residues. Both of these results indicate burial of some tryptophan residues of Hsp22 upon interaction with lipid vesicles. Membrane interactions also lead to increase in fluorescence polarization of Hsp22. Gel-filtration chromatography shows that Hsp22 binds stably with lipid vesicles; the extent of binding depends on the nature of the lipid. Hsp22 binds more strongly to vesicles made of lipids containing a phosphatidic acid, phosphatidylinositol or phosphatidylserine headgroup (known to be present in the inner leaflet of plasma membrane) compared with lipid vesicles made of a phosphatidylcholine head-group alone. Far-UV CD spectra reveal conformational changes upon binding to the lipid vesicles or in membrane-mimetic solvent, trifluoroethanol. Thus our fluorescence, CD and gel-filtration studies show that Hsp22 interacts with membrane and this interaction leads to stable binding and conformational changes. The present study therefore clearly demonstrates that Hsp22 exhibits potential membrane interaction that may play an important role in its cellular functions.     

Comparison of the interaction,positioning, structure induction and membrane perturbation of cell-penetrating peptides and non-translocating variants with phospholipid vesicles

Cell-penetrating peptides (CPPs) are able to translocate and carry cargo molecules across cell membranes. Using fluorescence techniques (polarization and quenching) and CD spectroscopy we studied the interaction, conformation and topology of two such peptides, transportan and 'penetratin' (pAntp), and two variants of differing translocating abilities, with small phospholipid vesicles of varying charge density. The induced structure of transportan is always helical independent of vesicle surface charge. pAntp and its two variants interact significantly only with negatively charged vesicles. The induced secondary structure depends on membrane charge and lipid/peptide ratio. The degree of membrane perturbation, evidenced by fluorescence polarization, of pAntp and its variants is related to their secondary structure. In the helical state, the peptides have little effect on the membrane. Under conditions where pAntp and its variants are converted into beta-structures, they cause membrane perturbation. Oriented CD suggests that the two CPPs (pAntp and transportan) in their helical state lie along the vesicle surface, while the two pAntp variants appear to penetrate deeper into the membrane.     

Tryptophan fluorescence study on the interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with model membranes

The interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with different phospholipid vesicles was investigated by fluorescence techniques, using a synthetic mutant signal peptide in which valine at position -8 in the hydrophobic sequence was replaced by tryptophan. First it was established that this mutation in the signal sequence of prePhoE does not affect in vivo and in vitro translocation efficiency and that the biophysical properties of the synthetic mutant signal peptide are similar to those of the wild-type signal peptide. Next, fluorescence experiments were performed which showed an increase in quantum yield and a blue shift of the emission wavelength maximum upon interaction of the signal peptide with lipid vesicles, indicating that the tryptophan moiety enters a more hydrophobic environment. These changes in intrinsic fluorescence were found to be more pronounced in the presence of phosphatidylglycerol (PG) or cardiolipin (CL) than with phosphatidylcholine (PC). In addition, quenching experiments demonstrated a shielding of the tryptophan fluorescence from quenching by the aqueous quenchers iodide and acrylamide upon interaction of the signal peptide with lipid vesicles, a shielding in the case of acrylamide that was more pronounced in the presence of negatively charged lipids. Finally it was found that acyl chain brominated lipids incorporated into phospholipid bilayers were able to quench the tryptophan fluorescence of the signal peptide, with the quenching efficiency in CL vesicles being much higher than in PC vesicles. The results clearly demonstrate that the PhoE signal peptide interacts strongly with different lipid vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence energy transfer between Ca2+ transport ATPase molecules in artificial membranes.

The purified ATPase of sarcoplasmic reticulum was covalently labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) or with iodoacetamidofluorescein (IAF). In reconstituted vesicles containing both types of ATPase molecules fluorescence energy transfer was observed from the IAEDANS (donor) to the IAF (acceptor) fluorophore as determined by the ratio of donor and acceptor fluorescence intensities, and by nanosecond decay measurements of donor fluorescence in the presence or absence of the acceptor. The observed energy transfer may arise by random collisions between ATPase molecules due to Brownian motion or by formation of complexes containing several ATPase molecules. Experimental distinction between these two models of energy transfer is possible based on predictions derived from mathematical models. Up to tenfold dilution of the lipid phase of reconstituted vesicles with egg lecithin had no measurable effect upon the energy transfer, suggesting that random collision between ATPase molecules in the lipid phase is not the principal cause of the observed effect. Addition of unlabeled ATPase in five- to tenfold molar excess over the labeled molecules abolished energy transfer. These observations together with electron microscopic and chemical cross-linking studies support the existence of ATPase oligomers in the membrane with sufficiently long lifetimes for energy transfer to occur. A hypothetical equilibrium between monomeric and tetrameric forms of the ATPase governed by the membrane potential is proposed as the structural basis of the regulation of Ca uptake and release by sarcoplasmic reticulum membranes during muscle contraction and relaxation.     

Brominated phospholipids as a tool for monitoring the membrane insertion of colicin A.

The intrinsic fluorescence of the colicin A thermolytic fragment does not change after insertion into normal phospholipid vesicles and is thus an unsuitable probe for monitoring the membrane insertion process. In this paper, we report the results of studies on the quenching of this fluorescence by brominated dioleoylphosphatidylglycerol (Br-DOPG) vesicles. Bromine atoms located at the midpoint of the phospholipid acyl chain quench the tryptophan fluorescence, indicating contact between fluorophores of the protein and the bilayer's hydrophobic core. Addition of Br-DOPG vesicles to a protein solution quenches the tryptophan fluorescence in a time-dependent manner. This quenching can be fitted to a single-exponential function, and thus interpreted as a one-step process. This allows calculation of an apparent rate constant of protein insertion into the membrane. Parameters known to affect the insertion of the thermolytic fragment into phospholipid monolayers or vesicles (pH and negative charge density) also affect the rate constant in comparable ways. In addition to the information gained concerning membrane exposure in the steady state, this approach provides the first real-time method for measuring the insertion of colicin into membranes. It is highly quantitative and can be used on all versions of the protein, e.g., full size, proteolytic fragments, and mutants. Brominated lipids provide experimental conditions identical to normal lipids and allow for great flexibility in protein/lipid ratios and concentrations. The kinetic analysis shows clearly the existence of a two-step process involving a rapid adsorption of the protein to the lipid surface followed by a slow insertion.     

Pressure tuning of the morphology of heterogeneous lipid vesicles: a two-photon-excitation fluorescence microscopy study

We used a technique that allows us to visualize local and morphological changes of the membrane of more component giant unilamellar vesicles due to high pressure perturbation. Under these conditions, thermally induced processes are largely suppressed, and the bending rigidity and line tension are influenced by pressure-induced changes in lipid molecular packing and shape only. We studied the effect of pressure on the lateral organization and morphology of the model raft system DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)/sphingomyelin/cholesterol as well as of the fluid mixture POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)/DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine) by two-photon excitation fluorescence microscopy. The pressure-dependent experiments were carried out using a sample cell made from a thin fused silica capillary. The use of Laurdan as fluorescence label allowed us to also follow the lipid phase state by calculating the generalized polarization (GP) values of the vesicles and extracting their average value. During the compression cycle, a reduction in the volume of the vesicles is observed, accompanied by an increase of the average GP value, indicating an increasingly tighter packing of the lipids. Interestingly, the two systems studied show phenomena of budding and fission, and these at surprisingly low pressures of 200-300 bar. Moreover, these budding processes are not directly related to phase transitions to an overall ordered conformational state of the lipid membrane, which occur at much higher pressures. The topological changes of the lipid vesicles are irreversible and exhibit a different behavior depending on whether the pressure is increased or decreased. The results are discussed in light of the various contributions to the free energy functional of lipid vesicles. Finally, the biological relevance of these studies is highlighted.     

Fluorescence study on the interaction of a multiple antigenic peptide from hepatitis A virus with lipid vesicles

The interaction of the multiple antigenic peptide MAP4VP3 with lipid membranes has been studied by spectroscopic techniques. MAP4VP3 is a multimeric peptide that corresponds to four units of the sequence 110-121 of the capsid protein VP3 of hepatitis A virus. In order to evaluate the electrostatic and hydrophobic components on the lipid-peptide interaction, small unilamelar vesicles of different compositions, including zwitterionic dipalmitoylphosphatidylcholine (DPPC), anionic dipalmitoylphosphatidylcholine/phatidylinositol (DPPC:PI 9:1), and cationic dipalmitoylphosphatidylcholine/stearylamine (DPPC:SA 9.5:0.5), were used as membrane models. Intrinsic tryptophan fluorescence changes and energy transfer experiments show that MAP4VP3 binds to all three types of vesicles with the same stoichiometry, indicating that the electrostatic component of the interaction is not important for binding of this anionic peptide. Steady-state polarization experiments with vesicles labeled with 1,6-diphenyl-1,3,5-hexatriene or with 1-anilino-8-naphtalene sulphonic acid indicate that MAP4VP3 induces a change in the packing of the bilayers, with a decrease in the fluidity of the lipids and an increase in the temperature of phase transition in all the vesicles. The percentage of lipid exposed to the bulk aqueous phase is around 60% in intact vesicles, and it does not change upon binding of MAP4VP3 to DPPC vesicles, indicating that the peptide does not alter the permeability of the membrane. An increase in the amount of lipid exposed to the aqueous phase in cationic vesicles indicates either lipid flip-flop or disruption of the vesicles. Binding to DPPC vesicles occurs without leakage of entrapped carboxyfluorescein, even at high mol fractions of peptide. However, a time-dependent leakage is seen with cationic DPPC/SA and anionic DPPC/PI vesicles, indicating that the peptide induces membrane destabilization and not lipid flip-flop. Resonance energy transfer experiments show that MAP4VP3 leakage from cationic vesicles is due to membrane fusion, whereas leakage from anionic vesicles is not accompanied by lipid mixing. Results show that MAP4VP3 interacts strongly with the lipid components of the membrane, and although binding is not of electrostatic nature, the bound form of the peptide has different activity depending on the membrane net charge; thus, it is membrane disruptive in cationic and anionic vesicles, whereas no destabilizing effect is seen in DPPC vesicles.     

Localization of intestinal sucrase-isomaltase complex on the microvillous membrane by electron microscopy using nonlabeled antibodies

Microvillous vesicles isolated from rabbit small intestine showed a trilaminar membrane with a rather smooth surface, which was apparently not affected by papain solubilizing sucrase-isomaltase complex or by trypsin unable to solubilize it. When microvilous vesicles or trysinized ones were incubated with immunoglobulin G against the sucrase-isomaltase complex or monovalent fragments therefrom, an apparently continuous electron-opaque layer approximately 180 A in width appeared around the external surface of vesicles. Such a layer was not formed on papainized vesicles. Microvillous vesicles and trypsinized ones negatively stained with phosphotungate showed a great number of particles protruding approximately 150 A from the membrane surface, but papainized vesicles did not. The particles existed close to one another and appeared to form a particulate layer 150 A in width on the surface. The antibodies, whether they were divalent or monovalent, increased the width of the layer to approximately 200 A and obscured the fine particulate structure of intact and trypsinized vesicles. Papainized vesicles retained their smooth surface upon interaction with antibodies. These results, together with those with the Triton-solubilized sucrase- isomaltase complex (Nishi and Takesue, 1978), J. Ultra-struct. Res., 62:1- 12), indicate not only that sucrase-isomaltase complexes are located close to one another on the membrane, but also that they or at least their protein portions protrude approximately 150 A from the surface of the trilaminar membrane.     

Osmotically induced fluid-phase uptake of fluorescent markers by protoplasts ofChenopodium album

Summary Protoplasts fromChenopodium album suspension culture show large, up to 5-fold, changes in surface area upon hypertonic or hypotonie treatment. These surface area variations cannot be explained by elastic stretching of the plasmalemma. An exchange of membrane material between the plasmalemma and an internal membrane source takes place. Fluid-phase uptake experiments with the fluorescence dyes 5, 6-carboxyfluorescein and Lucifer yellow CH demonstrated that osmotic shrinkage of protoplasts is accompanied by vesicular uptake of the external medium into protoplast cytoplasm. Confocal laser scanning microscopy, as well as conventional fluorescence microscopy, revealed the number, diameter and distribution of the osmocytotic vesicles at different osmotic levels. The rate of osmocytotic vesicle uptake was higher in the presence of calcium chloride than in the presence of EDTA in the external medium. At 6.9 mM calcium chloride we observed a loss of vesicular fluorescence upon returning protoplasts to 0.4 M from 0.8 M sorbitol.