Kinetics of membrane adhesion mediated by ligand-receptor interaction studied with a biomimetic system

We report the first measurement of the kinetics of adhesion of a single giant vesicle controlled by the competition between membrane-substrate interaction mediated by ligand-receptor interaction, gravitation, and Helfrich repulsion. To model the cell-tissue interaction, we doped the vesicles with lipid-coupled polymers (mimicking the glycocalix) and the reconstituted ligands selectively recognized by alpha(IIb)beta(3) integrin-mediating specific attraction forces. The integrin was grafted on glass substrates to act as a target cell. The adhesion of the vesicle membrane to the integrin-covered surface starts with the spontaneous formation of a small (approximately 200 nm) domain of tight adhesion, which then gradually grows until the whole adhesion area is in the state of tight adhesion. The time of adhesion varies from few tens of seconds to about one hour depending on the ligand and lipopolymer concentration. At small ligand concentrations, we observed the displacement xi of the front of tight adhesion following the square root law xi approximately t(1/2), whereas, at high concentrations, we found a linear law xi approximately t. We show both experimentally and theoretically that the t(1/2)-regime is dominated by diffusion of ligands, and the xi approximately t-regime by the kinetics of ligands-receptors association.     

Detection and analysis of membrane interactions by a biomimetic colorimetric lipid/polydiacetylene assay

We describe applications of a colorimetric assay based on supramolecular assemblies of lipid-polydiacetylene vesicles for analysis and screening of membrane interactions of lipophilic enzymes, peptides, and ions and for study of the effects of lipid composition upon membrane properties. The lipid-polymer aggregates undergo visible and quantifiable blue-to-red transitions following interfacial interactions and perturbation by varied biochemical processes. Specifically, we show that the colorimetric assay can be tuned for selective detection of enzymes reacting with different lipid species. The experiments also demonstrate that the lipid/polymer platform facilitates screening of peptide-membrane interactions in multicomponent mixtures. The colorimetric vesicles can incorporate lipid species from different cellular sources facilitating analysis of the contribution of molecular components to membrane properties and lipid interactions.     

Degradation of bacterial DNA by a natural antimicrobial agent with the help of biomimetic membrane system

The antimicrobial efficacy of methylglyoxal (MG) against several gram-negative bacteria including Escherichia coli has been reported. To determine the mechanism of action of MG, molecular interactions between lipid and MG within the liposomal membrane were also investigated. Multilamellar and unilamellar vesicles were prepared from 1, 2-dipalmitoyl-snglycero-3-phosphocholine (DPPC). The effect of MG on DPPC liposomal membrane was studied by fluorescence spectroscopy and differential scanning calorimetry. The results indicate that MG interacts mainly with the DPPC head group that produces a significant increase in the fluidity of liposomal vesicles, which could be the cause of a fusion/aggregation effect in microbial cells. The agarose gel electrophoresis study with the genomic DNA extracted from E. coli ATCC 25922 revealed that addition of MG could completely degrade this DNA within 1 h, pointing out to their distinctly high degree of sensitivity towards MG. Further, the drug was able to cross the cell membranes, penetrating into the interior of the cell and interacting with DNA for demonstrating antibacterial activity of MG.     

Pardaxin, a fish toxin peptide interaction with a biomimetic phospholipid/polydiacetylene membrane assay

Pardaxin is a fish toxin belonging to the alpha-helical, pore-forming peptide family, used in toxicological and biophysical research to study toxin-cell and -lipid-artificial membranes interactions. We investigated the membrane interaction of two pardaxin analogues using a colorimetric phospholipid/polydiacetylene biomimetic assay. In this assay, polydiacetylene undergoes visible, concentration dependent, blue-red transformation induced through interactions of pardaxins with the vesicle membrane. Pardaxins P4 and P5, are composed of 33 amino acids, but differ in a single amino acid substitution at the carboxy-terminal (G(31) to D(31), respectively) known to decrease the pore forming activity. Addition of pardaxins in the colorimetric assay induced dose-dependent color transitions with different kinetics. The colorimetric analysis could distinguish between different pardaxins-membrane interaction profiles, suggesting bilayer surface association for P4 and vesicle membrane penetration for P5. The colorimetric assay could distinguish between pardaxins membrane interaction profiles although circular dichroism spectra of vesicle-interacting pardaxins did not indicate a significant difference in the secondary structure between these two toxin analogues. The colorimetric platform utilized in the present report represents a useful assay with general applications for studying membrane interactions of peptides in general and pore-forming toxins in particular, and may become an important tool for evaluating quantitative toxin structure-activity relationship.     

Virus interactions with membrane filters

A survey of interactions of membrane filters with viruses has included 28 types of membranes, 4 types of enteroviruses, and 1 reovirus. Losses of these viruses in filtration, due to adsorption to the filter membranes, appear to be governed by three factors: the chemical composition of the filter membrane, the ratio of pore diameter to the diameter of the virus particle, and the presence of substances, such as those occurring in serum, which interfere with adsorption. Membranes of cellulose triacetate and of certain other materials have a very low affinity for these viruses. Cellulose triacetate filters adsorb virtually none when the pore size exceeds the virus diameter by a factor of more than 3. At porosities nearer the virus diameter, even low-affinity membranes adsorb large quantities of virus unless serum or some other additive interferes. Cellulose nitrate membranes, in the absence of interfering substances, adsorb enterovirus significantly at a pore size 285 times the virus diameter.     

Electronic wiring of a multi-redox site membrane protein in a biomimetic surface architecture

Bioelectronic coupling of multi-redox-site membrane proteins was accomplished with cytochrome c oxidase (CcO) as an example. A biomimetic membrane system was used for the oriented immobilization of the CcO oxidase on a metal electrode. When the protein is immobilized with the CcO binding side directed toward the electrode and reconstituted in situ into a lipid bilayer, it is addressable by direct electron transfer to the redox centers. Electron transfer to the enzyme via the spacer, referred to as electronic wiring, shows an exceptionally high rate constant. This allows a kinetic analysis of all four consecutive electron transfer steps within the enzyme to be carried out. Electron transfer followed by rapid scan cyclic voltametry in combination with surface-enhanced resonance Raman spectroscopy provides mechanistic and structural information about the heme centers. Probing the enzyme under turnover conditions showed mechanistic insights into proton translocation coupled to electron transfer. This bioelectronic approach opens a new field of activity to investigate complex processes in a wide variety of membrane proteins.     

Ganglioside GM1 forces the redistribution of cholesterol in a biomimetic membrane

Neutron reflectivity has been applied to investigate different mixed asymmetric lipid systems, in the form of single "supported+floating" bilayers, made of phospholipids, cholesterol and GM1 ganglioside (Neu5Acα2-3(Galβ1-3GalNAcβ1-4)Galβ1-4Glcβ1Cer)) in bio-similar mole ratios. Bilayer preparation was carried out layer-by-layer with the Langmuir-Blodgett Langmuir-Schaefer techniques, allowing for compositional asymmetry in the system buildup. It is the first time that such a complex model membrane system is reported. Two important conclusions are drawn. First, it is experimentally shown that the presence of GM1 enforces an asymmetry in cholesterol distribution, opposite to what happens for a GM1-free membrane that, submitted to a similar procedure, results in a full symmetrization of cholesterol distribution. We underline that natural cholesterol has been used. Second, and most interesting, our results suggest that a preferential asymmetric distribution of GM1 and cholesterol is attained in a model membrane with biomimetic composition, revealing that a true coupling between the two molecular species occurs.     

Stereostructure-based differences in the interactions of cardiotoxic local anesthetics with cholesterol-containing biomimetic membranes

Amide-type pipecoloxylidide local anesthetics, bupivacaine, and ropivacaine, show cardiotoxic effects with the potency depending on stereostructures. Cardiotoxic drugs not only bind to cardiomyocyte membrane channels to block them but also modify the physicochemical property of membrane lipid bilayers in which channels are embedded. The opposite configurations allow enantiomers to be discriminated by their enantiospecific interactions with another chiral molecule in membranes. We compared the interactions of local anesthetic stereoisomers with biomimetic membranes consisting of chiral lipid components, the differences of which might be indicative of the drug design for reducing cardiotoxicity. Fluorescent probe-labeled biomimetic membranes were prepared with cardiolipin and cholesterol of varying compositions and different phospholipids. Local anesthetics were reacted with the membrane preparations at a cardiotoxically relevant concentration of 200 μM. The potencies to interact with biomimetic membranes and change their fluidity were compared by measuring fluorescence polarization. All local anesthetics acted on lipid bilayers to increase membrane fluidity. Chiral cardiolipin was ineffective in discriminating S(-)-enantiomers from their antipodes. On the other hand, cholesterol produced the enantiospecific membrane interactions of bupivacaine and ropivacaine with increasing its composition in membranes. In 40 mol% and more cholesterol-containing membranes, the membrane-interacting potency was S(-)-bupivacaine相似文献   

Structure-activity relationships in the hydrophobic interactions of polyphenols with cellulose and collagen

Polyphenol interactions with both cellulose and collagen in the solid state have been studied by using chromatography on cellulose and by evaluating the hydrothermal stability of the polyphenol treated sheepskin collagen. Twenty-four polyphenolic compounds were studied, including seven glucose-based gallotannins, five polyalcohol-based gallotannins, and twelve ellagitannins. In the cellulose-polyphenols systems, the polyphenol's affinity to cellulose is positively correlated with their molecular masses, the number of galloyl groups, and their hydrophobicity (logP). The polyphenol treatment increased the hydrothermal stability of collagen samples, and such effects are also positively correlated with the molecular masses, total number of galloyl groups and the hydrophobicity of polyphenols. Ellagitannins showed much weaker interactions with both biopolymers than gallotannins having similar molecular mass, the same number of galloyl groups, and the same number of phenolic hydroxyl groups. It is concluded that, for the polyphenol interactions with both cellulose and collagen, (1) the galloyl group of polyphenols is the functional group; (2) the strength of interactions are positively correlated with molecular size, the number of galloyl groups and the hydrophobicity of polyphenols; (3) the hydrophobic interactions are of great significance; and (4) the interactions are strongly dependent on the flexibility of galloyl groups.     

Development of a biomimetic nanoporous membrane for the selective transport of charged proteins

We report the use of a micrometer-thick platinum-coated nanoporous membrane for the separation of differently charged proteins. A high field strength of about 25 kV m(-1) was applied, using very low transmembrane potentials of +/-1.5 V between the platinum-coated membranes. The system mimics the cell membrane function of facilitated transport for specific solutes. The selectivity for Lys:BSA:Mb in a mixed protein solution could be tuned readily between the flux ratios of 2:2:1 and 96:1:12 respectively, by simple variation of the transmembrane potentials from +1.5 V to -1.5 V. The experimental fluxes agreed closely with calculated fluxes derived from a simple electrophoresis-potential shielding model at favourable transmembrane potentials.     

A microfluidic lung-on-a-chip based on biomimetic hydrogel membrane

Lung-on-chips have showed great promise as a tool to recapitulate the respiratory system for investigation of lung diseases in the past decade. However, the commonly applied artificial elastic membrane (e.g., polydimethylsiloxane, PDMS) in the chip failed to mimic the alveolar basal membrane in the composition and mechanical properties. Here we replaced the PDMS film by a thin, biocompatible, soft, and stretchable membrane based on F127-DA hydrogel that well approached to the composition and stiffness of extracellular matrix in human alveoli for construction of lung-on-a-chip. This chip well reconstructed the mechanical microenvironments in alveoli so that the epithelial/endothelial functions were highly expressed with a well established alveolar-capillary barrier. In opposite to the unexpectedly accelerated fibrotic process on the PDMS-based lung-on-a-chip, HPAEpiCs on hydrogel-based chip only presented fibrosis under nonphysiologically high strain, well reflecting the features of pulmonary fibrosis in vivo. This physiologically relevant lung-on-a-chip would be an ideal model in investigation of lung diseases and for development of antifibrosis drugs.     

Cell-free production of VDAC directly into liposomes for integration with biomimetic membrane systems

The mitochondrial voltage-dependent anion channel (VDAC) is a pivotal protein since it provides the major transport pathway between the cytosol and the mitochondrial intermembrane space and it is implicated in cell apoptosis by functioning as a gatekeeper for the trafficking of mitochondrial death molecules. VDAC is a beta-barrel channel with a large conductance, and we use it as a model transport protein for the design of biomimetic systems. To overcome the limitations of classical overexpression methods for producing and purifying membrane proteins (MPs) we describe here the use of an optimized cell-free system. In a one-step reaction VDAC is obtained directly integrated into liposomes and purified by ultracentrifugation. We then combine proteoliposomes with different bilayers models in order to validate VDAC insertion and functionality. This VDAC biomimetic model is the first example validating the use of a cell-free expression system for production of MPs into liposomes and tethered bilayers as a toolbox to build a wide range of biomimetic devices.     

Structure-dependent effects of glucose-containing analogs of platelet activating factor (PAF) on membrane integrity

Synthetic choline-containing phospholipids comprise a new class of compounds with antineoplastic properties. We have investigated the effect of recently synthesized glucose-containing analogs of lysophosphatidylcholine (glyceroglucophospholipid, Glc-PC) and of lysoplatelet activating factor (Glc-PAF) and its C16, C14 and C12 derivatives (ET-16, ET-14, and ET-12) on proliferation of immortalized human keratinocyte (HaCaT) cells. The data were compared to the ability of the compounds to intercalate into phosphatidylserine liposomes and to form lesions in planar bilayer membranes. A correlation between bioactivity and membrane activity was found. The number of molecules that intercalated into phosphatidylserine liposomes depended on the chemical structure of the compounds and was in the order Glc-PAF approximately ET-16 approximately ET-14 > Glc-PC > ET-12. All compounds induced membrane lesions, and the lesion forming activity was in the same order. Similar activity rankings were found for the release of lactate dehydrogenase from HaCaT cells as a measure of lytic activity and for the influence on cell number as a measure of proliferation. In the latter test, however, proliferation was already inhibited at non-toxic concentrations. From these findings, it may be concluded that the intercalation of the compounds at toxic concentrations leads to the formation of membrane lesions and finally results in membrane rupture leading to cell death.     

The yeast split-ubiquitin system to study chloroplast membrane protein interactions

Each photosynthetic complex within the thylakoid membrane consists of several different subunits. During formation of these complexes, numerous regulatory factors are required for the coordinated transport and assembly of the subunits. Interactions between transport/assembly factors and their specific polypeptides occur in a membraneous environment and are usually transient and short-lived. Thus, a detailed analysis of the underlying molecular mechanisms by biochemical techniques is often difficult to perform. Here, we report on the suitability of a genetic system, i.e. the yeast split-ubiquitin system, to investigate protein–protein interactions of thylakoid membrane proteins. The data confirm the previously established binding of the cpSec-translocase subunits, cpSecY and cpSecE, and the interaction of the cpSec-translocase from Arabidopsis thaliana with Alb3, a factor required for the insertion of the light-harvesting chlorophyll-binding proteins into the thylakoid membrane. In addition, the proposed interaction between D1, the reaction center protein of photosystem II and the soluble periplasmic PratA factor from Synechocystis sp. PCC 6803 was verified. A more comprehensive analysis of Alb3-interacting proteins revealed that Alb3 is able to form dimers or oligomers. Interestingly, Alb3 was also shown to bind to the PSII proteins D1, D2 and CP43, to the PSI reaction center protein PSI-A and the ATP synthase subunit CF₀III, suggesting an important role of Alb3 in the assembly of photosynthetic thylakoid membrane complexes.     

CapZ-lipid membrane interactions: a computer analysis

Background  

CapZ is a calcium-insensitive and lipid-dependent actin filament capping protein, the main function of which is to regulate the assembly of the actin cytoskeleton. CapZ is associated with membranes in cells and it is generally assumed that this interaction is mediated by polyphosphoinositides (PPI) particularly PIP₂, which has been characterized in vitro.     

Spermine as a modulator of membrane fusion: interactions with acidic phospholipids

The interaction of spermine with acidic phospholipids was investigated for its possible relevance to membrane fusion. Equilibrium dialysis was used to measure the binding of spermine and calcium to large unilamellar vesicles (liposomes) of phosphatidate (PA) or phosphatidylserine (PS). Spermine bound to isolated PA and PS liposomes with intrinsic association constants of approximately 2 and 0.2 M-1, respectively. Above the aggregation threshold of the liposomes, the binding of spermine increased dramatically, especially for PA. The increased binding upon aggregation of PA liposomes was interpreted as evidence for the formation of a new binding complex after aggregation. Spermine enhanced calcium binding to PA, while it inhibited calcium binding to PS, under the same conditions. This difference explained the small effect of spermine on the overall rate of calcium-induced fusion of PS liposomes as opposed to the large effect on PA liposomes. The rate increase could be modeled by a spermine-induced increase in the liposome aggregation rate. The preference for binding of spermine to PA over PS suggested a preference for accessible monoesterified phosphate groups by spermine. This preference was confirmed by the large effects of spermine on aggregation and overall fusion rates of liposomes containing phosphatidylinositol 4,5-diphosphate. The large spermine effects on these liposomes compared with phosphatidate- or phosphatidylinositol-containing liposomes suggested that spermine has a strong specific interaction with phosphatidylinositol 4,5-diphosphate. Clearly, phosphorylation of phosphatidylinositol can lead to a large change in the spermine sensitivity of membrane fusion.