Interaction of the human N-Ras protein with lipid raft model membranes of varying degrees of complexity.

Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separation and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and functional aspects of three lipid raft model systems of varying degrees of biological relevance--PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol--using 2H solid-state nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the general behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composition of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras protein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-dependent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively simple models of raft membranes are able to reproduce many of their specific properties and functions.

High-fidelity protein targeting into membrane lipid microdomains in living cells.

Lipid analogues carrying three nitrilotriacetic acid (tris-NTA) head groups were developed for the selective targeting of His-tagged proteins into liquid ordered (lo ) or liquid disordered (ld ) lipid phases. Strong partitioning into the lo phase of His-tagged proteins bound to tris-NTA conjugated to saturated alkyl chains (tris-NTA DODA) was achieved, while tris-NTA conjugated to an unsaturated alkyl chain (tris-NTA SOA) predominantly resided in the ld phase. Interestingly, His-tag-mediated lipid crosslinking turned out to be required for efficient targeting into the lo phase by tris-NTA DODA. Robust partitioning into lo phases was confirmed by using viral lipid mixtures and giant plasma membrane vesicles. Moreover, efficient protein targeting into lo and ld domains within the plasma membrane of living cells was demonstrated by single-molecule tracking, thus establishing a highly generic approach for exploring lipid microdomains in situ.

An amphiphilic perylene imido diester for selective cellular imaging.

A new amphiphilic membrane marker based on a water-soluble dendritic polyglycerol perylene imido dialkylester has been designed, synthesized, and its optical properties characterized. In water it forms fluorescently quenched micellar self-aggregates, but when incorporated into a lipophilic environment, it monomerizes, and the highly fluorescent properties of the perylene core are recovered. These properties make it an ideal candidate for the imaging of artificial and cellular membranes as demonstrated by biophysical studies.

Structure and dynamics of molecular rods in membranes: application of a spin-labeled rod.

Molecular rods consisting of a hydrophobic backbone and terminally varying functional groups have been synthesized for applications for the functionalization of membranes. In the present study, we employ a spin-labeled analogue of a recently described new class of molecular rods to characterize their dynamic interactions with membranes. By using the different approaches of ESR and NMR spectroscopy, we show that the spin moiety of the membrane-embedded spin-labeled rod is localized in the upper chain/glycerol region of membranes of different compositions. The rod is embedded within the membrane in a tilted orientation to adjust for the varying hydrophobic thicknesses of these bilayers. This orientation does not perturb the membrane structure. The water solubility of the rod is increased significantly in the presence of certain cyclodextrins. These cyclodextrins also allow the rods to be extracted from the membrane and incorporated into preformed membranes. The latter will improve the future applications of these rods in cellular systems as stable membrane-associated anchors for the functionalization of membrane surfaces.

Cholesterol's aliphatic side chain modulates membrane properties.

The influence of cholesterol's alkyl side chain on membrane properties was studied using a series of synthetic cholesterol derivatives without a side chain or with a branched side chain consisting of 5 to 14 carbon atoms. Cholesterol's side chain is crucial for all membrane properties investigated and therefore essential for the membrane properties of eukaryotic cells.

New molecular rods--characterization of their interaction with membranes.

Molecular rods are synthetical molecules consisting of a hydrophobic backbone which are functionalized with varying terminal groups. Here, we report on the interaction of a recently described new class of molecular rods with lipid and biological membranes. In order to characterize this interaction, different fluorescently labeled rods were synthesized allowing for the application of fluorescence spectroscopy and microscopy based approaches. Our data show that the rods are incorporated into membranes with a perpendicular orientation to the membrane surface and enrich preferentially in liquid-disordered lipid domains. These characteristics underline that rods can be applied as stable membrane-associated anchors for functionalizing membrane surfaces.

Functional relevance of transmembrane domains in membrane fusion.

Membrane fusion is ubiquitous in life. Fusion of biological membranes is mediated by specialized fusion proteins anchored to the bilayers destined to fuse. Here we describe these proteins as being instrumental in viral, intracellular and developmental fusion. Next, we review experimental and theoretical evidence that points to fusion in the different systems as following a common 'fusion through hemifusion' pathway. We also focus on the structure and dynamics of the transmembrane segment that anchors the fusion proteins to the bilayer, and its role in driving fusion. In particular, we highlight the influence of this single segment on the surrounding membrane lipids and on the overall shape of the membrane along the way to fusion.

DBD dyes as fluorescent probes for sensing lipophilic environments.

Small fluorescent organic molecules based on [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) could be used as probes for lipophillic microenvironments in aqueous solutions by indicating the critical micelles concentration of detergents and staining cell organelles. Their fluorescence lifetime decreases drastically by the amount of water in their direct environment. Therefore they are potential probes for fluorescence lifetime imaging microscopy (FLIM).

Membrane-mediated induction and sorting of K-Ras microdomain signaling platforms.

The K-Ras4B GTPase is a major oncoprotein whose signaling activity depends on its correct localization to negatively charged subcellular membranes and nanoclustering in membrane microdomains. Selective localization and clustering are mediated by the polybasic farnesylated C-terminus of K-Ras4B, but the mechanisms and molecular determinants involved are largely unknown. In a combined chemical biological and biophysical approach we investigated the partitioning of semisynthetic fully functional lipidated K-Ras4B proteins into heterogeneous anionic model membranes and membranes composed of viral lipid extracts. Independent of GDP/GTP-loading, K-Ras4B is preferentially localized in liquid-disordered (l(d)) lipid domains and forms new protein-containing fluid domains that are recruiting multivalent acidic lipids by an effective, electrostatic lipid sorting mechanism. In addition, GDP-GTP exchange and, thereby, Ras activation results in a higher concentration of activated K-Ras4B in the nanoscale signaling platforms. Conversely, palmitoylated and farnesylated N-Ras proteins partition into the l(d) phase and concentrate at the l(d)/l(o) phase boundary of heterogeneous membranes. Next to the lipid anchor system, the results reveal an involvement of the G-domain in the membrane interaction process by determining minor but yet significant structural reorientations of the GDP/GTP-K-Ras4B proteins at lipid interfaces. A molecular mechanism for isoform-specific Ras signaling from separate membrane microdomains is postulated from the results of this study.

Direct visualization of large and protein-free hemifusion diaphragms.

Fusion of cellular membranes is a ubiquitous biological process requiring remodeling of two phospholipid bilayers. We believe it is very likely that merging of membranes proceeds via similar sequential intermediates. Contacting membranes form a stalk between the proximal leaflets that expands radially into an hemifusion diaphragm (HD) and subsequently open to a fusion pore. Although considered to be a key intermediate in fusion, direct experimental verification of this structure is difficult due to its transient nature. Using confocal fluorescence microscopy we have investigated the fusion of giant unilamellar vesicles (GUVs) containing phosphatidylserine and fluorescent virus derived transmembrane peptides or membrane proteins in the presence of divalent cations. Time-resolved imaging revealed that fusion was preceded by displacement of peptides and fluorescent lipid analogs from the GUV-GUV adhesion region. A detailed analysis of this area being several mum in size revealed that peptides were completely sequestered as expected for an HD. Lateral distribution of lipid analogs was consistent with formation of an HD but not with the presence of two adherent bilayers. Formation and size of the HD were dependent on lipid composition and peptide concentration.

Hemagglutinin of influenza virus partitions into the nonraft domain of model membranes.

The HA of influenza virus is a paradigm for a transmembrane protein thought to be associated with membrane-rafts, liquid-ordered like nanodomains of the plasma membrane enriched in cholesterol, glycosphingolipids, and saturated phospholipids. Due to their submicron size in cells, rafts can not be visualized directly and raft-association of HA was hitherto analyzed by indirect methods. In this study, we have used GUVs and GPMVs, showing liquid disordered and liquid ordered domains, to directly visualize partition of HA by fluorescence microscopy. We show that HA is exclusively (GUVs) or predominantly (GPMVs) present in the liquid disordered domain, regardless of whether authentic HA or domains containing its raft targeting signals were reconstituted into model membranes. The preferential partition of HA into ld domains and the difference between lo partition in GUV and GPMV are discussed with respect to differences in packaging of lipids in membranes of model systems and living cells suggesting that physical properties of lipid domains in biological membranes are tightly regulated by protein-lipid interactions.

Molecular rods with oligospiroketal backbones as anchors in biological membranes.

Getting stuck in: A hydrophobic molecular rod with terminal fluorescent moieties has been synthesized. The insertion of the rod into membranes was investigated and shown to incorporate efficiently into model and biological membranes (see picture; gray C, blue N, red O). Those rods can be used as stable membrane-associated anchors for functionalization of membrane surfaces.

Visualization of lipid domain-specific protein sorting in giant unilamellar vesicles.

Recent studies suggest that phospholipids in the plasma membrane of mammalian cells are not homogenously distributed but may form domains either by lipid-lipid interactions or/and as consequence of lipid-protein interactions. Such lipid compartments may act as protein recruiting platforms which, for example, are essential components of cell signaling pathways. Model membrane systems with a defined lipid composition are ideally suited to study domain-specific interactions of peripheral and integral membrane proteins. Giant unilamellar vesicles (GUVs) offer the opportunity to directly visualize in parallel, both the lateral lipid domains and the interaction sites of proteins using fluorescence microscopy. The application of GUVs is exemplarly illustrated for studying domain-specific interactions of the protein alpha-synuclein and the domain-specific distribution of synthetic transmembrane peptides.