On multivalent receptor activity of GM1 in cholesterol containing membranes.

Gangliosides located at the outer leaflet of plasma membrane are molecules that either participate in recognizing of exogenous ligand molecules or exhibit their own receptor activity, which are both essential phenomena for cell communication and signaling as well as for virus and toxin entry. Regulatory mechanisms of lipid-mediated recognition are primarily subjected to the physical status of the membrane in close vicinity of the receptor. Concerning the multivalent receptor activity of the ganglioside GM1, several regulatory strategies dealing with GM1 clustering and cholesterol involvement have been proposed. So far however, merely the isolated issues were addressed and no interplay between them investigated. In this work, several advanced fluorescence techniques such as Z-scan fluorescence correlation spectroscopy, Förster resonance energy transfer combined with Monte Carlo simulations, and a newly developed fluorescence antibunching assay were employed to give a more complex portrait of clustering and cholesterol involvement in multivalent ligand recognition of GM1. Our results indicate that membrane properties have an impact on a fraction of GM1 molecules that is not available for the ligand binding. While at low GM1 densities (~1 %) it is the cholesterol that turns GM1 headgroups invisible, at higher GM1 level (~4 %) it is purely the local density of GM1 molecules that inhibits the recognition. At medium GM1 content, cooperation of the two phenomena occurs. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.

GM1 Ganglioside Inhibits ß-Amyloid Oligomerization Induced by Sphingomyelin.

ß-Amyloid (Aß) oligomers are neurotoxic and implicated in Alzheimer's disease. Neuronal plasma membranes may mediate formation of Aß oligomers in vivo. Membrane components sphingomyelin and GM1 have been shown to promote aggregation of Aß; however, these studies were performed under extreme, non-physiological conditions. We demonstrate that physiological levels of GM1 , organized in nanodomains do not seed oligomerization of Aß40 monomers. We show that sphingomyelin triggers oligomerization of Aß40 and that GM1 is counteractive thus preventing oligomerization. We propose a molecular explanation that is supported by all-atom molecular dynamics simulations. The preventive role of GM1 in the oligomerization of Aß40 suggests that decreasing levels of GM1 in the brain, for example, due to aging, could reduce protection against Aß oligomerization and contribute to the onset of Alzheimer's disease.

Photoconversion of bodipy-labeled lipid analogues.

MOVING COLORS: Bodipy-labeled lipid analogues can change their photophysical properties and/or localization in the membrane upon light illumination. These changes are highly influenced by the lipid environment. This phenomenon can lead to lipid-environment-specific false positive signals in experimental techniques where spectral identity/separation is important.

Lipid Driven Nanodomains in Giant Lipid Vesicles are Fluid and Disordered.

It is a fundamental question in cell biology and biophysics whether sphingomyelin (SM)- and cholesterol (Chol)- driven nanodomains exist in living cells and in model membranes. Biophysical studies on model membranes revealed SM and Chol driven micrometer-sized liquid-ordered domains. Although the existence of such microdomains has not been proven for the plasma membrane, such lipid mixtures have been often used as a model system for 'rafts'. On the other hand, recent super resolution and single molecule results indicate that the plasma membrane might organize into nanocompartments. However, due to the limited resolution of those techniques their unambiguous characterization is still missing. In this work, a novel combination of Förster resonance energy transfer and Monte Carlo simulations (MC-FRET) identifies directly 10 nm large nanodomains in liquid-disordered model membranes composed of lipid mixtures containing SM and Chol. Combining MC-FRET with solid-state wide-line and high resolution magic angle spinning NMR as well as with fluorescence correlation spectroscopy we demonstrate that these nanodomains containing hundreds of lipid molecules are fluid and disordered. In terms of their size, fluidity, order and lifetime these nanodomains may represent a relevant model system for cellular membranes and are closely related to nanocompartments suggested to exist in cellular membranes.

Biophysical properties of cationic lipophosphoramidates: Vesicle morphology, bilayer hydration and dynamics.

Cationic lipids are used to deliver genetic material to living cells. Their proper biophysical characterization is needed in order to design and control this process. In the present work we characterize some properties of recently synthetized cationic lipophosphoramidates. The studied compounds share the same structure of their hydrophobic backbone, but differ in their hydrophilic cationic headgroup, which is formed by a trimethylammonium, a trimethylarsonium or a dicationic moiety. Dynamic light scattering and cryo-transmission electron microscopy proves that the studied lipophosphoramidates create stable unilamellar vesicles. Fluorescence of polarity probe, Laurdan, analyzed using time-dependent fluorescence shift method (TDFS) and generalized polarization (GP) gives important information about the phase, hydration and dynamics of the lipophosphoramidate bilayers. While all of the compounds produced lipid bilayers that were sufficiently fluid for their potential application in gene therapy, their polarity/hydration and mobility was lower than for the standard cationic lipid - DOTAP. Mixing cationic lipophosphoramidates with DOPC helps to reduce this difference. The structure of the cationic headgroup has an important and complex influence on bilayer hydration and mobility. Both TDFS and GP methods are suitable for the characterization of cationic amphiphiles and can be used for screening of the newly synthesized compounds.