Janja Majhenc

Myelin-like protrusions of giant phospholipid vesicles prepared by electroformation

Abstract Quasistable shapes of phospholipid bilayer vesicles obtained by formation in an alternating electric field [M.I. Angelova, S. Soleau, Ph. Meleard, J.F. Faucon, P. Bothorel, Prog. Colloid Polym. Sci. 89 (1992) 127; V. Heinrich, R.E. Waugh, Ann. Biomed. Eng. 24 (1996) 595] are observed. The vesicles appearing as composed of a mother sphere and a thin tubular myelin-like protrusion, are found to be a common phase in the spontaneous slow shape transformation that yields giant fluctuating phospholipid vesicles of different shapes. In the shape transformation, the myelin-like protrusion, which acts as a reservoir for the membrane area, is integrated into the mother vesicle.

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.

Mechanisms of equinatoxin II-induced transport through the membrane of a giant phospholipid vesicle.

Protein equinatoxin II from sea anemone Actinia equina L. was used to form pores in phospholipid membranes. We studied the effect of these pores on the net transmembrane transport of sucrose and glucose by observing single giant (cell-size) vesicles under the phase contrast microscope. Sugar composition in the vesicle was determined by measuring the width of the halo, which appears around the vesicle in the phase contrast image. The transport of sugars was induced when a vesicle, filled with the sucrose solution, was transferred into the isomolar environment of a glucose solution with added equinatoxin II. Typically, a vesicle grew to a critical size, then the membrane broke by bursting and the vesicle shrank, started to grow again, and the whole process was repeated. The consecutive membrane breaks occurred in the same spot. The observed behavior was interpreted by the diffusion flow of the glucose molecules through the equinatoxin II-induced pores and the consequent increase of the vesicle water content. The burst relaxed the critically strained membrane, which then apparently resealed. A mathematical model of the described behavior was developed and was used to obtain the equinatoxin II-induced membrane permeability for the glucose molecules. Its dependence on the equinatoxin II concentration is in agreement with the previous reports.

Cylindrical shapes of closed lipid bilayer structures correspond to an extreme area difference between the two monolayers of the bilayer.

The shapes of extreme area difference between the outer and the inner layer (deltaA) of the closed lipid bilayer structures at fixed membrane area (A) and fixed volume (V) are determined by stating and analytically solving a variational problem for axisymmetric shapes. It is shown that the spheres with at most two different radii and the cylinder are the solutions of this variational problem. The cylinder ended by a hemisphere on each end is the shape combined from these solutions and is therefore, itself the shape of the extreme deltaA at fixed V and A. The related cylindrical shapes of stearoyl-oleoyl-phosphocholine vesicles are shown.

Positioning of integrin β1, 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 β1, 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 β1 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 β1 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 β1 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.