Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Interfaculty Institute for Biochemistry

Max Planck Institute for Intelligent Systems

There is considerable interest in preparing cell-sized giant unilamellar vesicles from natural or nonnatural amphiphiles because a giant vesicle membrane resembles the self-closed lipid matrix of the plasma membrane of all biological cells. Currently, giant vesicles are applied to investigate certain aspects of biomembranes. Examples include lateral lipid heterogeneities, membrane budding and fission, activities of reconstituted membrane proteins, or membrane permeabilization caused by added chemical compounds. One of the challenging applications of giant vesicles include gene expressions inside the vesicles with the ultimate goal of constructing a dynamic artificial cell-like system that is endowed with all those essential features of living cells that distinguish them from the nonliving form of matter. Although this goal still seems to be far away and currently difficult to reach, it is expected that progress in this and other fields of giant vesicle research strongly depend on whether reliable methods for the reproducible preparation of giant vesicles are available. The key concepts of currently known methods for preparing giant unilamellar vesicles are summarized, and advantages and disadvantages of the main methods are compared and critically discussed.

Giant vesicles: preparations and applications.

Numerous experimental evidence show that exposure of biological systems to extremely high frequency microwaves may induce significant effects even at low powers. These effects are thought to occur via nonthermal mechanisms involving primarily the interaction of microwaves with phospholipid membrane structures. However, no conclusive experimental evidence that biomembranes exhibit remarkable sensitivity to this radiation has been provided up to now. Here, deuterium nuclear magnetic resonance spectroscopy is used to study the effects of microwaves on 1,2-Dimyristoyl-sn-glycero-3-phosphatidylcholine/(2)H(2)O multilamellar vesicles that serve as biomimetic membranes. Here we show that, if the membrane is brought into close proximity to the transition point, microwaves induce a reduction of water ordering at the membrane interface, an upward shift of the main phase transition temperature and a broadening of the transition region. A deep dosimetric analysis shows that the above effects are nonthermal, indicating the need for a nonthermal hypothesis to explain them. This study suggests that exposure to high-frequency microwaves can have far reaching consequences on active biological systems.

Microwave induced shift of the main phase transition in phosphatidylcholine membranes.

Exposure of cell membranes to an electromagnetic field (EMF) in the millimeter wave band (30-300 GHz) can produce a variety of responses. Further, many of the vibrational modes in complex biomolecules fall in the 1-100 GHz range. In addition to fundamental scientific interest, this may have applications in the development of diagnostic and therapeutic medical applications. In the present work, lipid vesicles of different size were used to study the effects of exposure to radiation at 52-72 GHz, with incident power densities (IPD) of 0.0035-0.010 mW/cm(2), on the chemical-physical properties of cell membranes. Large unilamellar vesicles (LUVs) were used to study the effect of the radiation on the physical stability of vesicles by dynamic light scattering. An inhibition of the aging processes (Ostwald ripening), which usually occur in these vesicles because of their thermodynamic instability, resulted. Giant unilamellar vesicles (GUVs) were used to study the effect of the radiation on membrane water permeability under osmotic stress by phase contrast microscopy. In this case, a decrease in the water membrane permeability of the irradiated samples was observed. We advance the hypothesis that both the above effects may be explained in terms of a change of the polarization states of water induced by the radiation, which causes a partial dehydration of the membrane and consequently a greater packing density (increased membrane rigidity).

The influence of millimeter waves on the physical properties of large and giant unilamellar vesicles.

Cardiolipin (CL) is a lipid with unique properties solely found in membranes generating electrochemical potential. It contains four acyl chains and tends to form nonlamellar structures, which are believed to play a key role in membrane structure and function. Indeed, CL alterations have been linked to disorders such as Barth syndrome and Parkinson's disease. However, the molecular effects of CL on membrane organization remain poorly understood. Here, we investigated the structure and physical properties of CL-containing membranes using confocal microscopy, fluorescence correlation spectroscopy, and atomic force microscopy. We found that the fluidity of the lipid bilayer increased and its mechanical stability decreased with CL concentration, indicating that CL decreases the packing of the membrane. Although the presence of up to 20% CL gave rise to flat, stable bilayers, the inclusion of 5% CL promoted the formation of flowerlike domains that grew with time. Surprisingly, we often observed two membrane-piercing events in atomic force spectroscopy experiments with CL-containing membranes. Similar behavior was observed with a lipid mixture mimicking the mitochondrial outer membrane composition. This suggests that CL promotes the formation of membrane areas with apposed double bilayers or nonlamellar structures, similar to those proposed for mitochondrial contact sites. All together, we show that CL induces membrane alterations that support the role of CL in facilitating bilayer structure remodeling, deformation, and permeabilization.

Cardiolipin effects on membrane structure and dynamics.

Besides their conventional role as energy suppliers for the cell, mitochondria in vertebrates are active regulators of apoptosis. They release apoptotic factors from the intermembrane space into the cytosol through a mechanism that involves the Bcl-2 protein family, mediating permeabilization of the outer mitochondrial membrane. Associated with this event, a number of additional changes affect mitochondria during apoptosis. They include loss of important mitochondrial functions, such as the ability to maintain calcium homeostasis and to generate ATP, as well as mitochondrial fragmentation and cristae remodeling. Moreover, the lipidic component of mitochondrial membranes undergoes important alterations in composition and distribution, which have turned out to be relevant regulatory events for the proteins involved in apoptotic mitochondrial damage.

Mitochondrial alterations in apoptosis.

The nonthermal biological effects of millimeter waves have been mainly attributed to the interaction with biological membranes. Several data on biomimetic membrane systems seem to support this conclusion. In this paper a mechanistic hypothesis is evaluated to explain such an interaction taking into account experimental NMR data on deuterium-labeled phospholipid vesicles. These data showed that millimeter waves induce a time and a hydration-dependent reduction of the water ordering around the phosphocholine headgroups. This effect is here interpreted as a change in membrane water partitioning, due to the coupling of the radiation with the fast rotational dynamics of bound water molecules, that results in a measurable relocation of water molecules from the inner to the outer binding regions of the membrane interface. When millimeter wave exposure is performed in the vicinity of the transition point, this effect can lead to an upward shift of the membrane phase transition temperature from the fluid to the gel phase. At a macroscopic level, this unique sensitivity may be explained by the universal dynamic behaviour of the membranes in the vicinity of the transition point, where a pretransitional increase of membrane area fluctuations, i.e., of the mean area per phospholipid headgroup, is observed. Exposure to millimeter waves increases the above fluctuations and enhances the second order character of the transition.

Katia Cosentino

Interfaculty Institute for Biochemistry

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers