Influence of stearyl and trifluoromethylquinoline modifications of the cell penetrating peptide TP10 on its interaction with a lipid membrane.

The PepFect family of cell-penetrating peptides (CPPs) was designed to improve the delivery of nucleic acids across plasma membranes. We present here a comparative study of two members of the family, PepFect3 (PF3) and PepFect6 (PF6), together with their parental CPP transportan-10 (TP10), and their interactions with lipid membranes. We show that the addition of a stearyl moiety to TP10 increases the amphipathicity of these molecules and their ability to insert into a lipid monolayer composed of zwitterionic phospholipids. The addition of negatively charged phospholipids into the monolayer results in decreased binding and insertion of the stearylated peptides, indicating modification in the balance of hydrophobic versus electrostatic interactions of peptides with lipid bilayer, thus revealing some clues for the selective interaction of these CPPs with different lipids. The trifluoromethylquinoline moieties, in PF6 make no significant contribution to membrane binding and insertion. TP10 actively introduces pores into the bilayers of large and giant unilamellar vesicles, while PF3 and PF6 do so only at higher concentrations. This is consistent with the lower toxicity of PF3 and PF6 observed in previous studies.

Positioning of integrin ß1, caveolin-1 and focal adhesion kinase on the adhered membrane of spreading cells.

We have investigated the relationship between the spreading of anchorage-dependent cells and the surface-density distribution of plasma membrane adhesion proteins. The surface positioning and density of integrin ß1, caveolin-1 (cav-1), the phosphorylated caveolin-1 (p-cav-1) and the focal adhesion kinase (FAK) located on the adhering cell membrane (ACM) of HUVEC cells was studied. Imaging with TIRF microscopy was used, which enabled us to observe a few-nanometers-thin section of the cell above the plasma membrane in combination with image-based analyses. Integrin ß1 and cav-1 have spatial interdependence on the ACM. Cells treated with substances that act on cell spreading caused changes in the size of the ACM area, as well as a redistribution of several proteins under investigation. Changes to the ACM area correlated positively with those to the surface density of the cav-1. The high integrin ß1 and the low cav-1 surface density, and vice versa, following the treatments show that the presence of one of them not only spatially excludes, but also reduces, the occurrence of the other protein on the ACM, which indicates a regulative mechanism between integrin ß1 and cav-1.

Macroscopic properties of phospholipid vesicles with a contact angle between the membrane domains.

Ternary mixtures of a high-melting lipid, a low-melting lipid, and cholesterol are known to form domains of a liquid-ordered and a liquid-disordered phase in bilayer membranes. We prepare giant vesicles from a sphingomyelin/dioleoylphosphocholine/cholesterol mixture and then examine them using fluorescence microscopy. NBD-labeled lipid and BODIPY-labeled cholesterol are used to identify the phase domains of the membrane. A vesicle with only two domains, one in a liquid-ordered and one in a liquid-disordered phase, is chosen because of its simple geometry, for convenient comparison of the experimental results with the theoretical predictions. A microinjector is used to gradually decrease and/or increase the volume of the vesicles by changing the osmolarity of the sugar solution. The relevant energy terms of the membrane mechanics are the elastic energies of the domains and the energy of the domain boundary. The elastic energy of the membrane domains can be described by two terms: the bending energy and the Gaussian bending energy. The energy of the domain boundary is proportional to its length. At the boundary between the domains a contact angle is taken into consideration. Then, in order to obtain values for the lateral tension and the contact angle, the areas of the domains and the characteristic dimensions of the shape are determined for different volumes. The best fits were obtained for a line tension of 6+/-3 pN and a contact angle of 1.4+/-0.3 rad.

Characteristics of phospholipid vesicles enhanced by adhesion on an annular region.

Phospholipid vesicle membranes are simple models used to study the mechanical properties of cell membranes. The shapes of flaccid vesicles can exhibit very diverse forms. When researching very flaccid vesicles, axisymmetrical vesicles with the membranes adhered to an annular region can also be observed. A phase diagram of such shapes was studied for different values of the vesicle parameters, i.e., the adhesion constant, the vesicle volume-to-membrane ratio, the volume ratio between the polar and the equatorial parts, and the equilibrium difference between the membrane monolayers. The energies of the annular shapes with respect to the vesicle parameters were closely examined and compared with the energies of the discocyte and stomatocyte shapes. The requirements for the existence of such annular shapes were also given for adhesion-free vesicle membranes. The results show that the adhesion between the lipid bilayers stabilizes the observed shapes, which belong to the locally stable branch of the annular vesicles. The value obtained for the adhesion constant of the SOPC membrane is 3×10^{-9}J/m^{2}.

The response of giant phospholipid vesicles to pore-forming peptide melittin.

The interaction between the pore-forming peptide melittin (MLT) and giant phospholipid vesicles was explored experimentally. Micromanipulation and direct optical observation of a vesicle (loaded with sucrose solution and suspended in isomolar glucose solution) enabled the monitoring of a single vesicle response to MLT. Time dependences of the vesicle size, shape and the composition of the inner solution were examined at each applied concentration of MLT (in the range from 1 to 60 microg/ml). The response varied with MLT concentration from slight perturbation of the membrane to disintegration of the vesicle. A model for MLT-vesicle interaction is proposed that explains the observed phenomena in the entire span of MLT concentrations and is consistent with deduced underlying mechanisms of MLT action: trans-membrane positioning and dimerization of MLT, the lipid flow from the outer to the inner membrane leaflet induced by MLT translocation, formation of pores and the consequent transport of small molecules through the membrane. The results of the theoretical analysis stress the role of dimers in the MLT-membrane interaction and demonstrate that the MLT-induced membrane permeability for sugar molecules in this experimental set-up depends on both MLT concentration and time.

Budding, vesiculation and permeabilization of phospholipid membranes-evidence for a feasible physiologic role of beta2-glycoprotein I and pathogenic actions of anti-beta2-glycoprotein I antibodies.

The in vivo physiologic role of beta2-glycoprotein I (beta2GPI) is presumed to be related to its interactions with negatively charged phospholipid membranes. Increased quantities of procoagulant microparticles derived by the vesiculation of blood cells have been detected in patients with antiphospholipid syndrome (APS) frequently associated with antibodies against beta2GPI (anti-beta2GPI). We investigated the influence of beta2GPI and anti-beta2GPI on giant phospholipid vesicles (GPVs). GPVs composed of phosphatidylserine and phosphatidylcholine were formed in an aqueous medium and individually transferred to a compartment containing either beta2GPI, anti-beta2GPI, or beta2GPI along with anti-beta2GPI. Shape changes of a single GPV were observed by a phase contrast microscope. Most GPVs transferred to the solution containing only beta2GPI budded moderately. Upon the transfer of GPVs to the solution containing beta2GPI and anti-beta2GPI either from patient with APS or mouse monoclonal anti-beta2GPI Cof-22, the budding was much more pronounced, generating also daughter vesicles. No such effects were seen when GPV was transferred to the solution containing anti-beta2GPI without beta2GPI. Our results suggest a significant physiologic role of beta2GPI in the budding of phospholipid membranes, which may be explained by the insertion of the C-terminal loop of beta2GPI into membranes, thus increasing the surface of the outer layer of a phospholipid bilayer. Anti-beta2GPI, recognizing domains I to IV of beta2GPI, enhanced the budding and vesiculation of GPVs in the presence of beta2GPI. This might be a novel pathogenic mechanism of anti-beta2GPI, promoting in vivo the expression of proadhesive and procoagulant phospholipid surfaces in APS.

Interaction of cetylpyridinium chloride with giant lipid vesicles.

The interaction of cationic surfactant cetylpyridinium chloride, CPC, with giant lipid vesicles prepared from 1-palmitoyl-2-oleoylphosphatidylcholine, POPC, was examined at various concentrations of the lipid component. The lipid concentration was determined by a spectrophotometric method. The potentiometric method based on surfactant-selective electrode was used for the determination of surfactant concentration in the external water solution. From these results, moles of surfactant incorporated in the membrane per mole of lipid (parameter beta) and two kinds of partition coefficients were calculated. Their values were found to be considerably larger than the available literature data. A three stage process of surfactant-induced solubilization of lipid vesicles was observed. First, stable mixed bilayers form, which become saturated with CPC at a value beta(sat) larger than 0.8, which then gradually disintegrate. Just prior to the breakdown of the vesicular structure, formation of ellipsoidal vesicles was observed by optical microscopy. This phenomenon was attributed to the cooperative incorporation of surfactant into the bilayer. Fluorescence measurements have shown that the second stage in the solubilization process of POPC by the C16 chain-length surfactant does not involve mixed micelles. These are formed only in the third stage, which is the complete solubilization of POPC bilayers. The corresponding critical micellization concentration decreases with increasing concentration of the lipid component.