Shedding light on membrane rafts structure and dynamics in living cells.

Cellular membranes are fundamental building blocks regulating an extensive repertoire of biological functions. These structures contain lipids and membrane proteins that are known to laterally self-aggregate in the plane of the membrane, forming defined membrane nanoscale domains essential for protein activity. Membrane rafts are described as heterogeneous, dynamic, and short-lived cholesterol- and sphingolipid-enriched membrane nanodomains (10-200 nm) induced by lipid-protein and lipid-lipid interactions. Those membrane nanodomains have been extensively characterized using model membranes and in silico methods. However, despite the development of advanced fluorescence microscopy techniques, undoubted nanoscale visualization by imaging techniques of membrane rafts in the membrane of unperturbed living cells is still uncompleted, increasing the skepticism about their existence. Here, we broadly review recent biochemical and microscopy techniques used to investigate membrane rafts in living cells and we enumerate persistent open questions to answer before unlocking the mystery of membrane rafts in living cells.

Super-Resolution Microscopy Using a Bioorthogonal-Based Cholesterol Probe Provides Unprecedented Capabilities for Imaging Nanoscale Lipid Heterogeneity in Living Cells.

Despite more than 20 years of work since the lipid raft concept was proposed, the existence of these nanostructures remains highly controversial due to the lack of noninvasive methods to investigate their native nanorganization in living unperturbed cells. There is an unmet need for probes for direct imaging of nanoscale membrane dynamics with high spatial and temporal resolution in living cells. In this paper, a bioorthogonal-based cholesterol probe (chol-N3 ) is developed that, combined with nanoscopy, becomes a new powerful method for direct visualization and characterization of lipid raft at unprecedented resolution in living cells. The chol-N3 probe mimics cholesterol in synthetic and cellular membranes without perturbation. When combined with live-cell super-resolution microscopy, chol-N3 demonstrates the existence of cholesterol-rich nanodomains of <50 nm at the plasma membrane of resting living cells. Using this tool, the lipid membrane structure of such subdiffraction limit domains is identified, and the nanoscale spatiotemporal organization of cholesterol in the plasma membrane of living cells reveals multiple cholesterol diffusion modes at different spatial localizations. Finally, imaging across thick organ samples outlines the potential of this new method to address essential biological questions that were previously beyond reach.

Molecular ruler mechanism and interfacial catalysis of the integral membrane acyltransferase PatA.

Glycolipids are prominent components of bacterial membranes that play critical roles not only in maintaining the structural integrity of the cell but also in modulating host-pathogen interactions. PatA is an essential acyltransferase involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. We demonstrate by electron spin resonance spectroscopy and surface plasmon resonance that PatA is an integral membrane acyltransferase tightly anchored to anionic lipid bilayers, using a two-helix structural motif and electrostatic interactions. PatA dictates the acyl chain composition of the glycolipid by using an acyl chain selectivity "ruler." We established this by a combination of structural biology, enzymatic activity, and binding measurements on chemically synthesized nonhydrolyzable acyl­coenzyme A (CoA) derivatives. We propose an interfacial catalytic mechanism that allows PatA to acylate hydrophobic PIMs anchored in the inner membrane of mycobacteria, through the use of water-soluble acyl-CoA donors.

Cholesterol in the Viral Membrane is a Molecular Switch Governing HIV-1 Env Clustering.

HIV-1 entry requires the redistribution of envelope glycoproteins (Env) into a cluster and the presence of cholesterol (chol) in the viral membrane. However, the molecular mechanisms underlying the specific role of chol in infectivity and the driving force behind Env clustering remain unknown. Here, gp41 is demonstrated to directly interact with chol in the viral membrane via residues 751-854 in the cytoplasmic tail (CT751-854). Super-resolution stimulated emission depletion (STED) nanoscopy analysis of Env distribution further demonstrates that both truncation of gp41 CT751-854 and depletion of chol leads to dispersion of Env clusters in the viral membrane and inhibition of virus entry. This work reveals a direct interaction of gp41 CT with chol and indicates that this interaction is an important orchestrator of Env clustering.

Lipidomimetic Compounds Act as HIV-1 Entry Inhibitors by Altering Viral Membrane Structure.

The envelope of Human Immunodeficiency Virus type 1 (HIV-1) consists of a liquid-ordered membrane enriched in raft lipids and containing the viral glycoproteins. Previous studies demonstrated that changes in viral membrane lipid composition affecting membrane structure or curvature can impair infectivity. Here, we describe novel antiviral compounds that were identified by screening compound libraries based on raft lipid-like scaffolds. Three distinct molecular structures were chosen for mode-of-action studies, a sterol derivative (J391B), a sphingosine derivative (J582C) and a long aliphatic chain derivative (IBS70). All three target the viral membrane and inhibit virus infectivity at the stage of fusion without perturbing virus stability or affecting virion-associated envelope glycoproteins. Their effect did not depend on the expressed envelope glycoproteins or a specific entry route, being equally strong in HIV pseudotypes carrying VSV-G or MLV-Env glycoproteins. Labeling with laurdan, a reporter of membrane order, revealed different membrane structure alterations upon compound treatment of HIV-1, which correlated with loss of infectivity. J582C and IBS70 decreased membrane order in distinctive ways, whereas J391B increased membrane order. The compounds' effects on membrane order were reproduced in liposomes generated from extracted HIV lipids and thus independent both of virion proteins and of membrane leaflet asymmetry. Remarkably, increase of membrane order by J391B required phosphatidylserine, a lipid enriched in the HIV envelope. Counterintuitively, mixtures of two compounds with opposite effects on membrane order, J582C and J391B, did not neutralize each other but synergistically inhibited HIV infection. Thus, altering membrane order, which can occur by different mechanisms, constitutes a novel antiviral mode of action that may be of general relevance for enveloped viruses and difficult to overcome by resistance development.

Pb(II) Induces Scramblase Activation and Ceramide-Domain Generation in Red Blood Cells.

The mechanisms of Pb(II) toxicity have been studied in human red blood cells using confocal microscopy, immunolabeling, fluorescence-activated cell sorting and atomic force microscopy. The process follows a sequence of events, starting with calcium entry, followed by potassium release, morphological change, generation of ceramide, lipid flip-flop and finally cell lysis. Clotrimazole blocks potassium channels and the whole process is inhibited. Immunolabeling reveals the generation of ceramide-enriched domains linked to a cell morphological change, while the use of a neutral sphingomyelinase inhibitor greatly delays the process after the morphological change, and lipid flip-flop is significantly reduced. These facts point to three major checkpoints in the process: first the upstream exchange of calcium and potassium, then ceramide domain formation, and finally the downstream scramblase activation necessary for cell lysis. In addition, partial non-cytotoxic cholesterol depletion of red blood cells accelerates the process as the morphological change occurs faster. Cholesterol could have a role in modulating the properties of the ceramide-enriched domains. This work is relevant in the context of cell death, heavy metal toxicity and sphingolipid signaling.

Structural Snapshots and Loop Dynamics along the Catalytic Cycle of Glycosyltransferase GpgS.

Glycosyltransferases (GTs) play a central role in nature. They catalyze the transfer of a sugar moiety to a broad range of acceptor substrates. GTs are highly selective enzymes, allowing the recognition of subtle structural differences in the sequences and stereochemistry of their sugar and acceptor substrates. We report here a series of structural snapshots of the reaction center of the retaining glucosyl-3-phosphoglycerate synthase (GpgS). During this sequence of events, we visualize how the enzyme guides the substrates into the reaction center where the glycosyl transfer reaction takes place, and unveil the mechanism of product release, involving multiple conformational changes not only in the substrates/products but also in the enzyme. The structural data are further complemented by metadynamics free-energy calculations, revealing how the equilibrium of loop conformations is modulated along these itineraries. The information reported here represent an important contribution for the understanding of GT enzymes at the molecular level.

Proteoliposomal formulations of an HIV-1 gp41-based miniprotein elicit a lipid-dependent immunodominant response overlapping the 2F5 binding motif.

The HIV-1 gp41 Membrane Proximal External Region (MPER) is recognized by broadly neutralizing antibodies and represents a promising vaccine target. However, MPER immunogenicity and antibody activity are influenced by membrane lipids. To evaluate lipid modulation of MPER immunogenicity, we generated a 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC)-based proteoliposome collection containing combinations of phosphatidylserine (PS), GM3 ganglioside, cholesterol (CHOL), sphingomyelin (SM) and the TLR4 agonist monophosphoryl lipid A (MPLA). A recombinant gp41-derived miniprotein (gp41-MinTT) exposing the MPER and a tetanus toxoid (TT) peptide that favors MHC-II presentation, was successfully incorporated into lipid mixtures (>85%). Immunization of mice with soluble gp41-MinTT exclusively induced responses against the TT peptide, while POPC proteoliposomes generated potent anti-gp41 IgG responses using lower protein doses. The combined addition of PS and GM3 or CHOL/SM to POPC liposomes greatly increased gp41 immunogenicity, which was further enhanced by the addition of MPLA. Responses generated by all proteoliposomes targeted the N-terminal moiety of MPER overlapping the 2F5 neutralizing epitope. Our data show that lipids impact both, the epitope targeted and the magnitude of the response to membrane-dependent antigens, helping to improve MPER-based lipid carriers. Moreover, the identification of immunodominant epitopes allows for the redesign of immunogens targeting MPER neutralizing determinants.

C8-glycosphingolipids preferentially insert into tumor cell membranes and promote chemotherapeutic drug uptake.

Insufficient drug delivery into tumor cells limits the therapeutic efficacy of chemotherapy. Co-delivery of liposome-encapsulated drug and synthetic short-chain glycosphingolipids (SC-GSLs) significantly improved drug bioavailability by enhancing intracellular drug uptake. Investigating the mechanisms underlying this SC-GSL-mediated drug uptake enhancement is the aim of this study. Fluorescence microscopy was used to visualize the cell membrane lipid transfer intracellular fate of fluorescently labeled C6-NBD-GalCer incorporated in liposomes in tumor and non-tumor cells. Additionally click chemistry was applied to image and quantify native SC-GSLs in tumor and non-tumor cell membranes. SC-GSL-mediated flip-flop was investigated in model membranes to confirm membrane-incorporation of SC-GSL and its effect on membrane remodeling. SC-GSL enriched liposomes containing doxorubicin (Dox) were incubated at 4°C and 37°C and intracellular drug uptake was studied in comparison to standard liposomes and free Dox. SC-GSL transfer to the cell membrane was independent of liposomal uptake and the majority of the transferred lipid remained in the plasma membrane. The transfer of SC-GSL was tumor cell-specific and induced membrane rearrangement as evidenced by a transbilayer flip-flop of pyrene-SM. However, pore formation was measured, as leakage of hydrophilic fluorescent probes was not observed. Moreover, drug uptake appeared to be mediated by SC-GSLs. SC-GSLs enhanced the interaction of doxorubicin (Dox) with the outer leaflet of the plasma membrane of tumor cells at 4°C. Our results demonstrate that SC-GSLs preferentially insert into tumor cell plasma membranes enhancing cell intrinsic capacity to translocate amphiphilic drugs such as Dox across the membrane via a biophysical process.

A cholesterol recognition motif in human phospholipid scramblase 1.

Human phospholipid scramblase 1 (SCR) catalyzes phospholipid transmembrane (flip-flop) motion. This protein is assumed to bind the membrane hydrophobic core through a transmembrane domain (TMD) as well as via covalently bound palmitoyl residues. Here, we explore the possible interaction of the SCR TMD with cholesterol by using a variety of experimental and computational biophysical approaches. Our findings indicate that SCR contains an amino acid segment at the C-terminal region that shows a remarkable affinity for cholesterol, although it lacks the CRAC sequence. Other 3-OH sterols, but not steroids lacking the 3-OH group, also bind this region of the protein. The newly identified cholesterol-binding region is located partly at the C-terminal portion of the TMD and partly in the first amino acid residues in the SCR C-terminal extracellular coil. This finding could be related to the previously described affinity of SCR for cholesterol-rich domains in membranes.

The C-terminal transmembrane domain of human phospholipid scramblase 1 is essential for the protein flip-flop activity and Ca²âº-binding.

Human phospholipid scramblase 1 (SCR) is a 318 amino acid protein that was originally described as catalyzing phospholipid transbilayer (flip-flop) motion in plasma membranes in a Ca²âº-dependent, ATP-independent way. Further studies have suggested an intranuclear role for this protein in addition. A putative transmembrane domain located at the C terminus (aa 291-309) has been related to the flip-flop catalysis. In order to clarify the role of the C-terminal region of SCR, a mutant was produced (SCRΔ) in which the last 28 amino acid residues were lacking, including the α-helix. SCRΔ had lost the scramblase activity and its affinity for Ca²âº was decreased by one order of magnitude. Fluorescence and IR spectroscopic studies revealed that the C-terminal region of SCR was essential for the proper folding of the protein. Moreover, it was found that Ca²âº exerted an overall destabilizing effect on SCR, which might facilitate its binding to membranes.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent oligomerization of fibroblast growth factor 2 (FGF2) triggers the formation of a lipidic membrane pore implicated in unconventional secretion.

Fibroblast growth factor 2 (FGF2) is a critical mitogen with a central role in specific steps of tumor-induced angiogenesis. It is known to be secreted by unconventional means bypassing the endoplasmic reticulum/Golgi-dependent secretory pathway. However, the mechanism of FGF2 membrane translocation into the extracellular space has remained elusive. Here, we show that phosphatidylinositol 4,5-bisphosphate-dependent membrane recruitment causes FGF2 to oligomerize, which in turn triggers the formation of a lipidic membrane pore with a putative toroidal structure. This process is strongly up-regulated by tyrosine phosphorylation of FGF2. Our findings explain key requirements of FGF2 secretion from living cells and suggest a novel self-sustained mechanism of protein translocation across membranes with a lipidic membrane pore being a transient translocation intermediate.

Sialyllactose in viral membrane gangliosides is a novel molecular recognition pattern for mature dendritic cell capture of HIV-1.

HIV-1 is internalized into mature dendritic cells (mDCs) via an as yet undefined mechanism with subsequent transfer of stored, infectious virus to CD4+ T lymphocytes. Thus, HIV-1 subverts a DC antigen capture mechanism to promote viral spread. Here, we show that gangliosides in the HIV-1 membrane are the key molecules for mDC uptake. HIV-1 virus-like particles and liposomes mimicking the HIV-1 lipid composition were shown to use a common internalization pathway and the same trafficking route within mDCs. Hence, these results demonstrate that gangliosides can act as viral attachment factors, in addition to their well known function as cellular receptors for certain viruses. Furthermore, the sialyllactose molecule present in specific gangliosides was identified as the determinant moiety for mDC HIV-1 uptake. Thus, sialyllactose represents a novel molecular recognition pattern for mDC capture, and may be crucial both for antigen presentation leading to immunity against pathogens and for succumbing to subversion by HIV-1.

Molecular recognition of a single sphingolipid species by a protein's transmembrane domain.

Functioning and processing of membrane proteins critically depend on the way their transmembrane segments are embedded in the membrane. Sphingolipids are structural components of membranes and can also act as intracellular second messengers. Not much is known of sphingolipids binding to transmembrane domains (TMDs) of proteins within the hydrophobic bilayer, and how this could affect protein function. Here we show a direct and highly specific interaction of exclusively one sphingomyelin species, SM 18, with the TMD of the COPI machinery protein p24 (ref. 2). Strikingly, the interaction depends on both the headgroup and the backbone of the sphingolipid, and on a signature sequence (VXXTLXXIY) within the TMD. Molecular dynamics simulations show a close interaction of SM 18 with the TMD. We suggest a role of SM 18 in regulating the equilibrium between an inactive monomeric and an active oligomeric state of the p24 protein, which in turn regulates COPI-dependent transport. Bioinformatic analyses predict that the signature sequence represents a conserved sphingolipid-binding cavity in a variety of mammalian membrane proteins. Thus, in addition to a function as second messengers, sphingolipids can act as cofactors to regulate the function of transmembrane proteins. Our discovery of an unprecedented specificity of interaction of a TMD with an individual sphingolipid species adds to our understanding of why biological membranes are assembled from such a large variety of different lipids.

Transbilayer (flip-flop) lipid motion and lipid scrambling in membranes.

This paper reviews the current knowledge on the various mechanisms for transbilayer, or flip-flop, lipid motion in model and cell membranes, enzyme-assisted lipid transfer by flippases, floppases and scramblases is briefly discussed, while non-catalyzed lipid flip-flop is reviewed in more detail. Transbilayer lipid motion may occur as a result of the insertion of foreign molecules (detergents, lipids, or even proteins) in one of the membrane leaflets. It may also be the result of the enzymatic generation of lipids, e.g. diacylglycerol or ceramide, at one side of the membrane. Transbilayer motion rates decrease in the order diacylglycerol>>ceramide>>phospholipids. Ceramide, but not diacylglycerol, can induce transbilayer motion of other lipids, and bilayer scrambling. Transbilayer lipid diffusion and bilayer scrambling are defined as two conceptually and mechanistically different processes. The mechanism of scrambling appears to be related to local instabilities caused by the non-lamellar ceramide molecule, or by other molecules that exhibit a relatively slow flip-flop rate, when asymmetrically inserted or generated in one of the monolayers in a cell or model membrane.

Determinants of specificity at the protein-lipid interface in membranes.

The complexity of pro- and eukaryotic lipidomes is increasingly appreciated mainly owing to the advance of mass spectrometric methods. Biophysical approaches have revealed that the large number of lipid classes and molecular species detected have implications for the self-organizing potential of biological membranes, resulting in the formation of lateral heterogeneous phases. How membrane proteins are able to adapt specifically to their surrounding heterogeneous matrix, and whether this environment affects protein targeting and function, is therefore a matter of particular interest. Here, we review specific protein-lipid interactions, focusing on the molecular mechanisms that determine specificity at the protein-lipid interface, and on membrane proteins that require lipids as cofactors for their architecture and function.

Ceramide-induced transbilayer (flip-flop) lipid movement in membranes.

Lipids in biological membranes are asymmetrically distributed across the bilayer. The choline-containing lipids, phosphatidylcholine (PtdCho) and sphingomyelin (SM), are more abundant in the external leaflet. In contrast, the amino-containing glycerophospholipids, phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEth), are located preferentially on the cytoplasmic leaflet. The maintenance of transbilayer lipid asymmetry is essential for normal membrane function, and disruption of this asymmetry is associated with cell activation or pathological condition. The physiological role of ceramide formation in response to cell stimulation remains controversial. Ceramide formation serves many different functions at various locations in the cell. Despite the limited capacity for spontaneous intracellular diffusion or membrane flip-flop of lipids in membranes, we have found that ceramide production, via sphingomyelinase action or addition of external ceramide, induces the transbilayer lipid motion of the lipids within the cellular membrane. This chapter outlines various commonly used assays for measuring lipid flip-flop induced by ceramide in cell and model membranes.

Protein-sphingolipid interactions within cellular membranes.

Each intracellular organelle critically depends on maintaining its specific lipid composition that in turn contributes to the biophysical properties of the membrane. With our knowledge increasing about the organization of membranes with defined microdomains of different lipid compositions, questions arise regarding the molecular mechanisms that underlie the targeting to/segregation from microdomains of a given protein. In addition to specific lipid-transmembrane segment interactions as a basis for partitioning, the presence in a given microdomain may alter the conformation of proteins and, thus, the activity and availability for regulatory modifications. However, for most proteins, the specific lipid environment of transmembrane segments as well as its relevance to protein function and overall membrane organization are largely unknown. To help fill this gap, we have synthesized a novel photoactive sphingolipid precursor that, together with a precursor for phosphoglycerolipids and with photo-cholesterol, was investigated in vivo with regard to specific protein transmembrane span-lipid interactions. As a proof of principle, we show specific labeling of the ceramide transporter with the sphingolipid probe and describe specific in vivo interactions of lipids with caveolin-1, phosphatidylinositol transfer protein beta, and the mature form of nicastrin. This novel photolabile sphingolipid probe allows the detection of protein-sphingolipid interactions within the membrane bilayer of living cells.

Sphingosine increases the permeability of model and cell membranes.

Sphingosine, at 5-15 mol % total lipids, remarkably increases the permeability to aqueous solutes of liposomal and erythrocyte ghost membranes. The increased permeability cannot be interpreted in terms of leakage occurring at the early stages of a putative membrane solubilization by sphingosine, nor is it due to a sphingosine-induced generation of nonlamellar structures, or flip-flop lipid movement. Instead, sphingosine stabilizes (rigidifies) gel domains in membranes, raising their melting temperatures and increasing the transition cooperativity. Structural defects originating during the lateral phase separation of the "more rigid" and "less rigid" domains are likely sites for the leakage of aqueous solutes to the extravesicular medium. The presence of coexisting domains in the plasma membrane makes it a target for sphingosine permeabilization. The sphingosine-induced increase in rigidity and breakdown of the plasma membrane permeability barrier could be responsible for some of the physiological effects of sphingosine.

Alkanes are not innocuous vehicles for hydrophobic reagents in membrane studies.

Alkanes (C6-C16) are often used as vehicles for hydrophobic reagents, e.g. long-chain ceramides, in cell biology studies. It is generally understood that they are inert solvents, particularly when added in small volumes. However, simple calculations show that, under standard experimental conditions in cell studies, alkane: phospholipid molar ratios in excess of 1000:1 may be found. Even at much smaller ratios (close to 1:1) our studies with liposomes show that alkanes induce vesicle aggregation. Differential scanning calorimetry shows marked changes in both the gel-fluid and the lamellar-hexagonal transitions. Alkanes inhibit bacterial sphingomyelinase when acting on large unilamellar vesicles, and activate bacterial phospholipase C under the same conditions. Thus, the use of alkanes in cell studies requires strict control experiments to avoid artefactual results.