Gangliosides are important glycosphingolipids involved in a multitude of physiological functions. From a physicochemical standpoint, this is related to their ability to self-organize into nanoscopic domains, even at molar concentrations of one per 1000 lipid molecules. Despite recent experimental and theoretical efforts suggesting that a hydrogen bonding network is crucial for nanodomain stability, the specific ganglioside moiety decisive for the development of these nanodomains has not yet been identified. Here, we combine an experimental technique achieving nanometer resolution (Förster resonance energy transfer analyzed by Monte Carlo simulations) with atomistic molecular dynamic simulations to demonstrate that the sialic acid (Sia) residue(s) at the oligosaccharide headgroup dominates the hydrogen bonding network between gangliosides, driving the formation of nanodomains even in the absence of cholesterol or sphingomyelin. Consequently, the clustering pattern of asialoGM1, a Sia-depleted glycosphingolipid bearing three glyco moieties, is more similar to that of structurally distant sphingomyelin than that of the closely related gangliosides GM1 and GD1a with one and two Sia groups, respectively.

• MeSH
• G(M1) Ganglioside MeSH
• Gangliosides * chemistry   MeSH
• Glycosphingolipids MeSH
• Sphingomyelins * MeSH
• Molecular Dynamics Simulation MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• G(M1) Ganglioside MeSH
• Gangliosides * MeSH
• Glycosphingolipids MeSH
• Sphingomyelins * MeSH

Plasma membranes as well as their simplified model systems show an inherent nanoscale heterogeneity. As a result of strong interleaflet interactions, these nanoheterogeneities (called here lipid nanodomains) can be found in perfect registration (i.e., nanodomains in the inner leaflet are registered with the nanodomains in the outer leaflet). Alternatively, they might be interleaflet independent, antiregistered, or located asymmetrically in one bilayer leaflet only. To distinguish these scenarios from each other appears to be an experimental challenge. In this work, we analyzed the potential of Förster resonance energy transfer to characterize interleaflet organization of nanodomains. We generated in silico time-resolved fluorescence decays for a large set of virtual as well as real donor/acceptor pairs distributed over the bilayer containing registered, independent, antiregistered, or asymmetrically distributed nanodomains. In this way, we were able to identify conditions that gave satisfactory or unsatisfactory resolution. Overall, Förster resonance energy transfer appears as a robust method that, when using donor/acceptor pairs with good characteristics, yields otherwise difficult-to-reach characteristics of membrane lipid nanodomains.

• MeSH
• Models, Biological MeSH
• Cell Membrane metabolism   MeSH
• Lipid Bilayers metabolism   MeSH
• Membrane Lipids * MeSH
• Membranes metabolism   MeSH
• Fluorescence Resonance Energy Transfer * methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Lipid Bilayers MeSH
• Membrane Lipids * MeSH

The plasma membrane is a complex system, consisting of two layers of lipids and proteins compartmentalized into small structures called nanodomains. Despite the asymmetric composition of both leaflets, coupling between the layers is surprisingly strong. This can be evidenced, for example, by recent experimental studies performed on phospholipid giant unilamellar vesicles showing that nanodomains formed in the outer layer are perfectly registered with those in the inner leaflet. Similarly, microscopic phase separation in one leaflet can induce phase separation in the opposing leaflet that would otherwise be homogeneous. In this review, we summarize the current theoretical and experimental knowledge that led to the current view that domains are - irrespective of their size - commonly registered across the bilayer. Mechanisms inducing registration of nanodomains suggested by theory and calculations are discussed. Furthermore, domain coupling is evidenced by experimental studies based on the sparse number of methods that can resolve registered from independent nanodomains. Finally, implications that those findings using model membrane studies might have for cellular membranes are discussed.

• Keywords
• biomembranes, domain registration, interleaflet coupling, membrane asymmetry, nanodomains, phase separation, plasma membranes,
• Publication type
• Journal Article MeSH
• Review MeSH

Many membrane proteins are thought to function as dimers or higher oligomers, but measuring membrane protein oligomerization in lipid membranes is particularly challenging. Förster resonance energy transfer (FRET) and fluorescence cross-correlation spectroscopy are noninvasive, optical methods of choice that have been applied to the analysis of dimerization of single-spanning membrane proteins. However, the effects inherent to such two-dimensional systems, such as the excluded volume of polytopic transmembrane proteins, proximity FRET, and rotational diffusion of fluorophore dipoles, complicate interpretation of FRET data and have not been typically accounted for. Here, using FRET and fluorescence cross-correlation spectroscopy, we introduce a method to measure surface protein density and to estimate the apparent Förster radius, and we use Monte Carlo simulations of the FRET data to account for the proximity FRET effect occurring in confined two-dimensional environments. We then use FRET to analyze the dimerization of human rhomboid protease RHBDL2 in giant plasma membrane vesicles. We find no evidence for stable oligomers of RHBDL2 in giant plasma membrane vesicles of human cells even at concentrations that highly exceed endogenous expression levels. This indicates that the rhomboid transmembrane core is intrinsically monomeric. Our findings will find use in the application of FRET and fluorescence correlation spectroscopy for the analysis of oligomerization of transmembrane proteins in cell-derived lipid membranes.

• MeSH
• Cell Membrane metabolism   MeSH
• Dimerization MeSH
• Humans MeSH
• Membrane Lipids metabolism   MeSH
• Membrane Proteins * metabolism   MeSH
• Protein Multimerization MeSH
• Fluorescence Resonance Energy Transfer * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Lipids MeSH
• Membrane Proteins * MeSH

The dynamics of cellular membranes is primarily determined by lipid species forming a bilayer. Proteins are considered mainly as effector molecules of diverse cellular processes. In addition to large assemblies of proteins, which were found to influence properties of fluid membranes, biological membranes are densely populated by small, highly mobile proteins. However, little is known about the effect of such proteins on the dynamics of membranes. Using synthetic peptides, we demonstrate that transmembrane helices interfere with the mobility of membrane components by trapping lipid acyl chains on their rough surfaces. The effect is more pronounced in the presence of cholesterol, which segregates from the rough surface of helical peptides. This may contribute to the formation or stabilization of membrane heterogeneities. Since roughness is a general property of helical transmembrane segments, our results suggest that, independent of their size or cytoskeleton linkage, integral membrane proteins affect local membrane dynamics and organization.

• Keywords
• Biophysics, Computational Molecular Modelling, Membrane Architecture, Protein Physics,
• Publication type
• Journal Article MeSH

In this perspective we summarize current knowledge of the effect of monosialoganglioside GM1 on the membrane-mediated aggregation of the β-amyloid (Aβ) peptide. GM1 has been suggested to be actively involved in the development of Alzheimer's disease due to its ability to seed the aggregation of Aβ. However, GM1 is known to be neuroprotective against Aβ-induced toxicity. Here we suggest that the two scenarios are not mutually exclusive but rather complementary, and might depend on the organization of GM1 in membranes. Improving our understanding of the molecular details behind the role of gangliosides in neurodegenerative amyloidoses might help in developing disease-modifying treatments.

• MeSH
• Amyloid beta-Peptides chemistry   metabolism   MeSH
• G(M1) Ganglioside chemistry   metabolism   MeSH
• Humans MeSH
• Brain metabolism   MeSH
• Protein Aggregation, Pathological metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Amyloid beta-Peptides MeSH
• G(M1) Ganglioside MeSH

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.

• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Fluorescence methods are versatile tools for obtaining dynamic and topological information about biomembranes because the molecular interactions taking place in lipid membranes frequently occur on the same timescale as fluorescence emission. The fluorescence intensity decay, in particular, is a powerful reporter of the molecular environment of a fluorophore. The fluorescence lifetime can be sensitive to the local polarity, hydration, viscosity, and/or presence of fluorescence quenchers/energy acceptors within several nanometers of the vicinity of a fluorophore. Illustrative examples of how time-resolved fluorescence measurements can provide more valuable and detailed information about a system than the time-integrated (steady-state) approach will be presented in this review: 1), determination of membrane polarity and mobility using time-dependent spectral shifts; 2), identification of submicroscopic domains by fluorescence lifetime imaging microscopy; 3), elucidation of membrane leakage mechanisms from dye self-quenching assays; and 4), evaluation of nanodomain sizes by time-resolved Förster resonance energy transfer measurements.

• MeSH
• Fluorescent Dyes chemistry   MeSH
• Microscopy, Fluorescence methods   MeSH
• Kinetics MeSH
• Lipid Bilayers chemistry   MeSH
• Fluorescence Resonance Energy Transfer methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Fluorescent Dyes MeSH
• Lipid Bilayers MeSH

The formation of membrane heterogeneities, e.g., lipid domains and pores, leads to a redistribution of donor (D) and acceptor (A) molecules according to their affinity to the structures formed and the remaining bilayer. If such changes sufficiently influence the Förster resonance energy transfer (FRET) efficiency, these changes can be further analyzed in terms of nanodomain/pore size. This paper is a continuation of previous work on this theme. In particular, it is demonstrated how FRET experiments should be planned and how data should be analyzed in order to achieve the best possible resolution. The limiting resolution of domains and pores are discussed simultaneously, in order to enable direct comparison. It appears that choice of suitable donor/acceptor pairs is the most crucial step in the design of experiments. For instance, it is recommended to use DA pairs, which exhibit an increased affinity to pores (i.e., partition coefficients K(D,A) > 10) for the determination of pore sizes with radii comparable to the Förster radius R(0). On the other hand, donors and acceptors exhibiting a high affinity to different phases are better suited for the determination of domain sizes. The experimental setup where donors and acceptors are excluded from the domains/pores should be avoided.

• MeSH
• Fluorescent Dyes chemistry   pharmacokinetics   MeSH
• Ion Channels chemistry   metabolism   MeSH
• Lipid Bilayers chemistry   metabolism   MeSH
• Membrane Microdomains chemistry   metabolism   MeSH
• Monte Carlo Method MeSH
• Fluorescence Resonance Energy Transfer * MeSH
• Models, Theoretical MeSH
• Tissue Distribution MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fluorescent Dyes MeSH
• Ion Channels MeSH
• Lipid Bilayers MeSH