Behavior of GM3 ganglioside in lipid monolayers mimicking rafts or fluid phase in membranes

We studied the interaction of GM3 ganglioside with sphingomyelin (SM) and palmitoyl-oleoyl-phosphatidylcholine (POPC) in Langmuir monolayers mimicking, respectively, raft and fluid phase of a cellular membrane, by surface pressure measurements and fluorescence microscopy. No difference was observed in the behavior of SM-GM3 and POPC-GM3 monolayers. In both cases, a GM3 threshold concentration has been underlined between 20 and 40 mol%. Below this threshold, SM-GM3 and POPC-GM3 monolayers behave ideally, suggesting that GM3 and host lipid would form separated domains. On the contrary, above the threshold, a condensation of monolayers is observed. This could be due to a partial solubilisation of GM3 in host lipid, leading to a change in orientation of GM3 molecules at the air-water interface.     

Gelation of gelatin observation in the bulk and at the air-water interface

Gelation of gelatin under various conditions has been followed by atomic force microscopy (AFM) with the objective of understanding more fully the structure formed during the gelation process. AFM images were obtained of the structures formed from both the bulk sol and in surface films during the onset of gelation. While gelation occurred in the bulk sol, the extent of helix formation was monitored by measurements of optical rotation, and the molecular aggregation was imaged by AFM. Interfacial gelatin films formed at the air-water interface were also studied. Measurements of surface tension and surface rheology were made periodically and Langmuir-Blodgett films were drawn from the interface to allow AFM imaging of the structure of the interfacial layer as a function of time. Structural studies reveal that at low levels of helical content the gelatin molecules assemble into aggregates containing short segments of dimensions comparable to those expected for gelatin triple helices. With time larger fibrous structures appear whose dimensions suggest that they are bundles of triple helices. As gelation proceeds, the number density of fibers increases at the expense of the smaller aggregates, eventually assembling into a fibrous network. The gel structure appears to be sensitive to the thermal history, and this is particularly important in determining the structure and properties of the interfacial films. © 1998 John Wiley & Sons, Inc. Biopoly 46: 245–252, 1998     

Antitrypanosomal activity of epi-polygodial from Drimys brasiliensis and its effects in cellular membrane models at the air-water interface

Epi-polygodial, a drimane sesquiterpene was isolated from Drimys brasiliensis (Winteraceae). This compound demonstrated high parasite selectivity towards Trypanosoma cruzi trypomastigotes (IC₅₀ = 5.01 μM) with a selectivity index higher than 40. These results were correlated with the effects observed when this compound was incorporated in cellular membrane models of protozoans, represented by Langmuir monolayers of dipalmitoylphosphoethanolamine (DPPE). Surface pressure-area isotherms showed that epi-polygodial expands DPPE monolayers at higher areas and condenses them at lower areas, which was attributed to the preferential interaction with the polar heads of the lipid. This mechanism of action could be corroborated with Polarization-Modulation Reflection-Absorption Spectroscopy and Brewster Angle Microscopy. These results pointed to the fact that the interaction of epi-polygodial with DPPE monolayers at the air-water interface affects the physical chemical properties of the mixed film, which may be important to comprehend the interaction of this drug with cellular membranes at the molecular level.     

Enzymatic generation of ceramide induces membrane restructuring: Correlated AFM and fluorescence imaging of supported bilayers

The effect of enzymatic generation of ceramide on phase separated bilayers with a mixture of co-existing fluid and liquid-ordered phases has been examined using a combination of atomic force microscopy (AFM) and fluorescence imaging. Supported lipid bilayers prepared from a DOPC/sphingomyelin/cholesterol mixture were imaged prior to, during and after incubation with sphingomyelinase by total internal reflection fluorescence (TIRF) microscopy. Enzyme treatment resulted in the growth of large dye-excluded regions. The growth kinetics for these patches are consistent with activity of a variable number of enzyme molecules in different regions of the bilayer. Correlated AFM and fluorescence imaging shows that some of the large dye-excluded patches form around the original liquid-ordered domains, which become heterogeneous in height with many raised ceramide-rich regions around their periphery. However, some of the dye-excluded patches correspond to areas of the bilayer where the initial domains have largely or partially disappeared. The dye-excluded patches observed by fluorescence are shown to be areas of increased adhesion in lateral deflection AFM images and are postulated to form by incorporation of both cholesterol and ceramide in the original fluid phase and to vary in composition throughout the bilayer. This is evident from the observation that the dye-excluded areas are all detected as areas of increased friction, but do not always show a distinct height difference in topographic images. These results highlight the utility of a multi-modal imaging approach for understanding the complex membrane restructuring that occurs upon enzymatic generation of ceramide.     

Spread mixed monolayers of deoxycholic and dehydrocholic acids at the air-water interface, effect of subphase pH. Characterization by axisymmetric drop shape analysis

Bile acids (deoxycholic and dehydrocholic acids) spread mixed monolayers behavior at the air/water interface were studied as a function of subphase pH using a constant surface pressure penetration Langmuir balance based on the Axisymmetric Drop Shape Analysis (ADSA). We examined the influence of electrostatic, hydrophobic and hydration forces on the interaction between amphiphilic molecules at the interface by the collapse area values, the thermodynamic parameters and equation of state virial coefficients analysis. The obtained results showed that at neutral (pH=6.7) or basic (pH=10) subphase conditions the collapse areas values are similar to that of cholanoic acid and consistent with the cross-sectional area of the steroid nucleus (approximately 40 A(2)). The Gibbs energy of mixing values (DeltaG(mix)<0) and the first virial coefficients of the equation of state (b(0)<1) indicated that a miscible monolayer with laterally structured microdomains existed. The aggregation number (1/b(0)) was estimated within the order of 6 (pH=6.7) and 3 (pH=10). At pH=3.2, acidic subphase conditions, no phase separation occurs (DeltaG(mix)<0) but a high expanded effect of the monolayer could be noted. The mixed monolayer behavior was no ideal and no aggregates were formed (b(0)> or =1). Such behavior indicates that the polar groups of the molecules interacts each other more strongly by repulsive electrostatic forces than with the more hydrophobic part of the molecule.     

Erythrocyte death in vitro induced by starvation in the absence of Ca

Human erythrocytes (RBCs), stored at 4 °C under nominal absence of external energy sources and calcium ions, show a gradual decrease in membrane roughness (R_rms) at the end of which the appearance of morphological phenomena (spicules, vesicles and spherocytes) is observed on the cell membrane, phenomena that can mainly be ascribed to the ATP-dependent disconnection of the cortical cytoskeleton from the lipid bilayer. After depletion of the intracellular energy sources obtained under the extreme conditions chosen, treatment with a minimal rejuvenation solution makes the following remarks possible: (i) RBCs are able to regenerate adenosine triphosphate (ATP) and 2,3-bisphosphoglycerate only up to 4 days of storage at 4 °C, whereas from the eighth day energy stocks cannot be replenished because of a disorder in the transmembrane mechanisms of transport; (ii) the RBCs' roughness may be restored to the initial value (i.e. that observed in fresh RBCs) only in samples stored up to 4-5 days, whereas after the eighth day of storage the rejuvenation procedure appears to be inefficient; (iii) membrane physical properties - as measured by R_rms - are actually controlled by the metabolic production of ATP, necessary to perform the RBCs' basic functions; (iv) once energy stores cannot be replenished, a regulated sequence of the morphological events (represented by local buckles that lead to formation of spicules and vesicles of the lipid bilayer with generation of spherocytes) is reminiscent of the RBCs' apoptotic final stages; (v) the morphological phenomenology of the final apoptotic stages is passive (i.e. determined by simple mechanical forces) and encoded in the mechanical properties of the membrane-skeleton; and (vi) necrotic aspects (e.g. disruption of cell membrane integrity, so that intracellular protein content is easily released) ensue when RBCs are almost totally (≥ 90%) depleted in an irreversible way of the energetic stores.     

Compositional domain immiscibility in whole myelin monolayers at the air-water interface and Langmuir-Blodgett films

Monomolecular layers of whole myelin membrane can be formed at the air-water interface from vesicles or from solvent solution of myelin. The films appear microheterogeneous as seen by epifluorescence and Brewster angle microscopy. The pattern consists mainly of two coexisting liquid phases over the whole compression isotherm. The liquid nature of the phases is apparent from the fluorescent probe behavior, domain mobility, deformability and boundary relaxation due to the line tension of the surface domains. The monolayers were transferred to alkylated glass and fluorescently labeled against myelin components. The immunolabeling of two major proteins of myelin (myelin basic protein, proteolipid-DM20) and of 2′,3′-cyclic nucleotide 3′-phosphodiesterase shows colocalization with probes partitioning preferentially in liquid-expanded lipid domains also containing ganglioside G_M1. A different phase showing an enrichment in cholesterol, galactocerebroside and phosphatidylserine markers is also found. The distribution of components is qualitatively independent of the lateral surface pressure and is generally constituted by one phase enriched in charged components in an expanded state coexisting with another phase enriched in non-charged constituents of lower compressibility. The domain immiscibility provides a physical basis for the microheterogeneity found in this membrane model system.     

Study of calix[4]resorcinarene-dopamine complexation in mixed phospholipid monolayers formed at the air-water interface

We have studied the physical properties of monolayers formed by calix[4]resorcinarene and in mixtures with dipalmitoyl phosphatidylcholine (DPPC) in various molar ratios formed at the air-water interface and at presence of dopamine in water subphase by means of measurements of surface pressure and dipole potential. We showed that both calix[4]resorcinarene as well as its mixture with DPPC form stable monolayers at the water subphase. The presence of dopamine resulted in an increase of the mean molecular area and in a decrease of the compressibility modulus of the monolayers. For mixed monolayers at higher content of calix[4]resorcinarene (> 0.2 molar fraction) a deviation from ideal miscibility took place especially for monolayers in a solid state. This can be connected with formation of aggregates of calix[4] resorcinarene. Lowest miscibility and weakest interaction of dopamine with a monolayer was observed for calix[4]resorcinarene molar fraction of 0.33 in the monolayer.     

Roughness of the plasma membrane as an independent morphological parameter to study RBCs: A quantitative atomic force microscopy investigation

A novel approach to the study of RBCs based on the collection of three-dimensional high-resolution AFM images and on the measure of the surface roughness of their plasma membrane is presented. The dependence of the roughness from several parameters of the imaging was investigated and a general rule for a trustful analysis and comparison has been suggested. The roughness of RBCs is a morphology-related parameter which has been shown to be characteristic of the single cells composing a sample, but independent of the overall geometric shape (discocyte or spherocyte) of the erythrocytes, thus providing extra-information with respect to a conventional morphology study. The use of the average roughness value as a label of a whole sample was tested on different kinds of samples. Analyzed data revealed that the quantitative roughness value does not change after treatment of RBCs with various commonly used fixation and staining methods while a drastic decrease occurs when studying cells with membrane-skeletal alteration both naturally occurring or artificially induced by chemical treatments. The present method provides a quantitative and powerful tool for a novel approach to the study of erythrocytes structure through an ultrastructural morphological analysis with the potential to give information, in a non-invasive way, on the RBCs function.     

Interfacial behavior of cytoplasmic and mitochondrial creatine kinase oligomeric states

Adsorption to the air/water interface of isoenzymes of creatine kinase was investigated using surface pressure-area isotherms and Brewster angle microscopy (BAM) observations. Octameric mitochondrial creatine kinase (mtCK) exhibits a significant affinity for the air/water interface. Whatever the mode of formation of the interfacial film, i.e., injection of the protein in the subphase or spreading onto the buffer surface, the final arrangement and conformation adopted by mtCK molecules lead to a similar result. In contrast, the dimeric isoenzymes mtCK and cytosolic MMCK do not induce any surface pressure variation. However, when the subphase contains 0.3M NaCl, both isoenzymes adsorb to the interface. When treated with 0.8 or 3M GdnHCl, muscle creatine kinase (MMCK) becomes surface active and occupies a greater surface than mtCK. This result contrasts with previous observations, often derived from monomeric proteins, that their surface activity is increased upon unfolding. It underlines the possible influence exerted by the protein oligomeric state on its interfacial activity. At a subphase pH of 8.8, which corresponds to the pI of octameric mtCK, the profiles of the isotherms obtained with dimeric and octameric states and the resistance to compression of the protein monolayers are significantly affected when compared to those recorded at pH 7.4. These data suggest that the octamer is more hydrophobic than the dimer and may contribute to explaining why octamers bind to the inner mitochondrial membrane while dimers do not.     

Structural and functional properties of irradiated nuclear membranes at the air-water phase interface

The monolayer technique, the methods of electron microscopy and IR-spectroscopy were used to study isolated nuclear membranes of calf thymus cells. The data obtained permitted to study the relationship between structural disorders induced by irradiation of membranes, the changes in their functional status, and the role played by a lipid component of membranes in these processes.     

Surface behavior,microheterogeneity and adsorption equilibrium of myelin at the air-water interface

Interfacial films of whole myelin membrane adsorb at the air-water interface from myelin vesicles. The films show a liquid state and their equilibrium spreading pressure is equal to the collapse pressure (about 47 mN/m). The films appear microheterogeneous as seen by epifluorescence microscopy, consisting in two liquid phases over all the adsorption isotherm, starting with rounded liquid expanded domains (low surface pressure) immersed in a cholesterol enriched phase and reaching a fractal pattern at high surface pressure similar to those previously observed by compressing the film. Vesicles adsorb to the interfacial film mainly at the lateral interfaces. The high surface pressure at equilibrium (almost equal to the collapse pressure) indicates the formation of surface multilayers, also shown by fluorescence microscopy.     

Cholesterol favors the anchorage of human dystrophin repeats 16 to 21 in membrane at physiological surface pressure

Dystrophin (DYS) is a filamentous protein that connects the cytoskeleton and the extracellular matrix via the sarcolemma, conferring resistance to muscular cells. In this study, interactions between the DYS R16–21 fragment and lipids were examined using Langmuir films made of anionic and zwitterionic lipids. The film fluidity was modified by the addition of 15% cholesterol. Whatever the lipid mixture examined, at low surface pressure (20 mN/m) few differences appeared on the protein insertion and the presence of cholesterol did not affect the protein/lipid interactions. At high surface pressure (30 mN/m), the protein insertion was very low and occurred only in zwitterionic films in the liquid-expanded phase. In anionic films, electrostatic interactions prevented the protein insertion outright, and caused accumulation of the protein on the hydrophilic part of the monolayer. Addition of cholesterol to both lipid mixtures drastically modified the protein–lipid interactions: the DYS R16–21 insertion increased and its organization in the monolayer appeared to be more homogeneous. The presence of accessible cholesterol recognition amino-acid consensus sequences in this fragment may enhance the protein/membrane binding at physiological lateral pressure. These results suggest that the anchorage of dystrophin to the membrane in vivo may be stabilized by cholesterol-rich nano-domains in the inner leaflet of sarcolemma.     

Solution of fatty acids from monolayers spread at the air-water interface: identification of phase transformations and the estimation of surface charge

The contraction or decrease in area of fatty acid monolayers maintained at a constant surface pressure of 16 dynes/cm was studied as a function of fatty acid chain length, unsaturation, temperature, and the hydrogen ion concentration in the subphase. The data were consistent with the hypothesis that fatty acid solution from the monolayer into the subphase was the mechanism for film loss. Autoxidative reactions did not contribute significantly to film loss since contraction occurred with saturated fatty acid monolayers and with unsaturated fatty acid monolayers in an anaerobic environment. The decrease in area per unit time or the solution rate was inversely proportional to chain length and directly proportional to the degree of unsaturation. Arrhenius plots showed activation energies of 1.5-2.5 kcal mole(-1) for tetradecanoic, octadecenoic, and octadecadienoic acids, and 25 kcal mole(-1) for hexadecanoic acid. The solution rate from the monolayer increased in a sigmoidal fashion with an increase in subphase pH, and the apparent surface pK(a) was estimated as the point where the solution rate was half-maximum. Apparent surface pK(a) values were: hexadecanoic acid, 9.7; octadecenoic acid, 8.3; tetradecanoic acid, 7.9; and octadecadienoic acid, 8.0.     

Microscopy basics and the study of actin–actin-binding protein interactions

Actin is a multifunctional eukaryotic protein with a globular monomer form that polymerizes into a thin, linear microfilament in cells. Through interactions with various actin-binding proteins (ABPs), actin plays an active role in many cellular processes, such as cell motility and structure. Microscopy techniques are powerful tools for determining the role and mechanism of actin–ABP interactions in these processes. In this article, we describe the basic concepts of fluorescent speckle microscopy, total internal reflection fluorescence microscopy, atomic force microscopy, and cryoelectron microscopy and review recent studies that utilize these techniques to visualize the binding of actin with ABPs.     

Interaction of proteins with lipid monolayers at the air-solution interface studied by reflection spectroscopy

The interaction of a fluorescein-labelled insulin and of cytochrome C with the air-solution interface and with lipid monolayers at the air-solution interface has been studied by measuring the change in surface pressure at constant area and by reflection spectroscopy. Chromophores at the interface only give rise to enhanced light reflection without contribution to the signal from chromophores in the bulk. The accumulation of labelled insulin at the solution surface is very weak as concluded from the shape of the spectrum and reflection intensity. No interaction with a monolayer of dipalmitoyl-phosphatidylcholine at initial surface pressure of 5mN/m was detected. In contrast, the interaction with monolayers of dioctadecyl-dimethyl-ammonium bromide at initial surface pressures between 5 and 40 mN/m is much stronger, leading to a remarkable increase of surface pressure at constant area and strong reflection signal. The technique was also used to detect cytochrome C at the air-solution interface.     

Effect of the covalently linked fatty acid 18-MEA on the nanotribology of hair's outermost surface

Highly ordered lipids adsorbed or grafted on surfaces are known to provide protection and lubrication custom engineered surfaces. We have used atomic force microscopy (AFM) to measure adhesion and frictional properties of the outermost surfaces of a variety of human hairs with the aim of both understanding the role of 18-methyleicosanoic acid (18-MEA), an unusual branched-chain fatty acid covalently bound to the cuticle surface, and investigating how treatments or the ethnic origin affect this layer. Results show that an unmodified silicon nitride AFM tip is able to detect changes at the hair surface that can be related to the absence or presence of this layer due to treatment conditions and in particular that this monolayer has a lubricant effect.     

Molecular organization of surfactin-phospholipid monolayers: Effect of phospholipid chain length and polar head

Mixed monolayers of the surface-active lipopeptide surfactin-C₁₅ and various lipids differing by their chain length (DMPC, DPPC, DSPC) and polar headgroup (DPPC, DPPE, DPPS) were investigated by atomic force microscopy (AFM) in combination with molecular modeling (Hypermatrix procedure) and surface pressure-area isotherms. In the presence of surfactin, AFM topographic images showed phase separation for each surfactin-phospholipid system except for surfactin-DMPC, which was in good agreement with compression isotherms. On the basis of domain shape and line tension theory, we conclude that the miscibility between surfactin and phospholipids is higher for shorter chain lengths (DMPC > DPPC > DSPC) and that the polar headgroup of phospholipids influences the miscibility of surfactin in the order DPPC > DPPE > DPPS. Molecular modeling data show that mixing surfactin and DPPC has a destabilizing effect on DPPC monolayer while it has a stabilizing effect towards DPPE and DPPS molecular interactions. Our results provide valuable information on the activity mechanism of surfactin and may be useful for the design of surfactin delivery systems.     

Hyaluronan conformations on surfaces: effect of surface charge and hydrophobicity

Extended, relaxed, condensed, and interacting forms of the polysaccharide hyaluronan have been observed by atomic force microscopy (AFM). The types of images obtained depend on the properties of the surfaces used. We have investigated several different surface conditions for HA imaging, including unmodified mica, mica chemically modified with two different kinds of amino-terminated silanes (3-aminopropyltriethoxysilane and N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride), and highly oriented pyrolytic graphite. We found the degree of HA molecular extension or condensation to be variable, and the number of bound chains per unit area was low, for all of the mica-based surfaces. HA was more easily imaged on graphite, a hydrophobic surface. Chains were frequently observed in high degrees of extension, maintained by favorable interaction with the surface after molecular combing. This observation suggests that the HA macromolecule interacts with graphite through hydrophobic patches along its surface. AFM studies of HA behavior on differing surfaces under well-controlled environmental conditions provides useful insight into the variety of conformations and interactions likely to be found under differing physiological conditions.