Gregor Gomišček

Microtubes and nanotubes of a phospholipid bilayer membrane

We propose a theory describing the stable structure of a phospholipid bilayer in pure water involving a spherical mother vesicle with long thin tubular protrusion. It is considered that the phospholipid molecules are in general anisotropic with respect to the axis normal to the membrane and can orient in the plane of the membrane if the curvature field is strongly anisotropic. Taking this into account, the membrane free energy is derived starting from a single-molecule energy and using methods of statistical mechanics. By linking the description on the microscopic level with the continuum theory of elasticity we recover the expression for the membrane bending energy and obtain an additional (deviatoric) contribution due to the orientational ordering of the phospholipid molecules. It is shown that the deviatoric contribution may considerably decrease the phospholipid vesicle membrane free energy if the vesicle involves regions where the difference between the two principal curvatures is large (thin cylindrical protrusions and/or thin finite necks) and thereby yields a possible explanation for the stability of the long thin tubular protrusions of the phospholipid bilayer vesicles. We report on the experiment exhibiting a stable shape of the spherical phospholipid vesicle with a long thin tubular protrusion in pure water.

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.

Effects of the pore-forming agent nystatin on giant phospholipid vesicles.

The effects of the polyene pore-forming agent nystatin were investigated on individual giant unilamellar phospholipid vesicles (GUVs), made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), in different methanol-water solutions using phase-contrast optical microscopy. Three characteristic effects were detected in three different nystatin concentration ranges: vesicle shape changes (between 150 and 250μM); transient, nonspecific, tension pores (between 250 and 400μM); and vesicle ruptures (above 400μM). Both the appearance of the transient tension pores and the vesicle ruptures were explained as being a consequence of the formation of size-selective nystatin channels, whose membrane area density increases with the increasing nystatin concentrations. Our results also show that nystatin is able to form pores in the absence of sterols. In addition, study of the cross-interactions between nystatin and methanol revealed mutually antagonizing effects on the vesicle behavior for methanol volume fractions higher than 10%.

The role of sterols in the lipid vesicle response induced by the pore-forming agent nystatin.

The influences of ergosterol and cholesterol on the activity of the nystatin were investigated experimentally in a POPC model membrane as well as theoretically. The behavior of giant unilamellar vesicles (GUVs) under osmotic stress due to the formation of transmembrane pores was observed on single vesicles at different nystatin concentrations using phase-contrast microscopy. A significant shift of the typical vesicle behavior, i.e., morphological alterations, membrane bursts, slow vesicle ruptures and explosions, towards lower nystatin concentrations was detected in the ergosterol-containing vesicles and a slight shift towards higher nystatin concentrations was detected in the cholesterol-containing membranes. In addition, the nystatin activity was shown to be significantly affected by the ergosterol membranes molar fraction in a non-proportional manner. The observed tension-pore behavior was interpreted using a theoretical model based on the osmotic phenomena induced by the occurrence of size-selective nystatin pores. The number of nystatin pores for different vesicle behavior was theoretically determined and the role of the different mechanical characteristics of the membrane, i.e., the membranes expansivity and bending moduli, the line tension and the lysis tension, in the tension-pore formation process was quantified. The sterol-induced changes could not be explained adequately on the basis of the different mechanical characteristics, and were therefore interpreted mainly by the direct influences of the membrane sterols on the membrane binding, the partition and the pore-formation process of nystatin.

The influence of very low illumination on the postural sway of young and elderly adults.

The purpose of the present study was to evaluate the influence of very low ambient illumination and complete darkness on the postural sway of young and elderly adults. Eighteen healthy young participants aged 23.8±1.5 years and 26 community-dwelling elderly aged 69.8±5.6 years were studied. Each participant performed four tests while standing on a force platform in the following conditions: in normal light (215 lx) with open eyes and with closed eyes, in very low illumination (0.25 lx) with open eyes, and in complete darkness with open eyes. The sequences of the tests in the altered visual conditions were determined by random blocs. Postural sway was assessed by means of the force platform measurements. The centre of pressure variables: the medio-lateral and antero-posterior path lengths, mean velocities, sway areas, and fractal dimensions were analysed. Very low illumination resulted in a statistically significant increase in postural sway in both the young and elderly groups compared to normal light, although the increase was significantly smaller than those observed in the eyes closed and complete darkness condition, and no significant effects of illumination on fractal dimensions were detected. The gains of the sways in the very low or no illumination conditions relative to the normal light condition were significantly larger in the group of young participants than in the group of elderly participants (up to 50% and 25%, respectively). However, the response patterns to changes in illumination were similar in the young and elderly participants, with the exception of the short-range fractal dimension of the medio-lateral sway. In conclusion, very low illumination resulted in increased postural sway compared to normal illumination; however, in the closed eye and complete darkness conditions, postural sway was significantly higher than in the very low illumination condition regardless of the age of the participants.

Rotation of giant phospholipid vesicles in an uniform shear flow.

Rotation of giant “point attached” phospholipid (POPC) vesicles in a shear flow was studied. The dependence of the angular velocity on the flow gradient was measured and the experimental results were compared to the predictions of a theoretical model. A good linear correlation between the angular velocity of the vesicle and the flow gradient, as predicted, was observed.

Osmotic Effects Induced by Pore-Forming Agent Nystatin: From Lipid Vesicles to the Cell

The responses of Chinese hamster ovary epithelial cells, caused by the pore-forming agent nystatin, were investigated using brightfield and fluorescence microscopy. Different phenomena, i.e., the detachment of cells, the formation of blebs, the occurrence of “cell-vesicles” and cell ruptures, were observed. These phenomena were compared to those discovered in giant lipid vesicles. A theoretical model, based on the osmotic effects that occur due to the size-discriminating nystatin transmembrane pores in lipid vesicles, was extended with a term that considers the conservation of the electric charge density in order to describe the cell’s behavior. The increase of the cellular volume was predicted and correlated with the observed phenomena.

Magnetic resonance, a phenomenon with a great potential in medicine, but with a complex physical background - Part 3: The basic principles of magnetic resonance imaging.

Two basic problems have to be solved when dealing with imaging techniques – the adequate image contrast and the spatial reconstruction of images. In this article, the basic concepts of solving these two problems in the case of magnetic resonance imaging (MRI) are shown. Based on the spin echo, three ways of contrast optimization, i.e., spin density, T1- and T2-weighted MRI, are dealt with more thoroughly. The use of contrast media is also discussed. Furthermore, we explain the basic method of spatial encoding in MRI, i.e., the determination of the location of tissue signal source with the use of magnetic field gradients (“the principle of frequency encoding”), and the use of the Fourier transform. Finally, the back-projection reconstruction technique is presented.

Asymmetrical labeling of giant phospholipid vesicles

Abstract The method for labeling of inner membrane leaflet in unilamellar giant POPC vesicles was developed and characterised. Symmetrically NBD-PC labeled vesicles were treated by sodium dithionite, which undergoes an irreversible chemical reaction with NBD-PC molecule making it non-fluorescent. After the addition of dithionite the fluorescence on single vesicles as well as on vesicle suspension showed a 50 % decrease of its initial value corresponding to marker quenching in the outer leaflet. Hence, flourimetry as well as flourescence microscopy prove that dithionite quenching is a suitable method to induce an asymmetrical labeling of the NBD-PC marked giant vesicles.