Raft Bilayers Research Articles

Hydrolysis of sphingomyelin in supported raft bilayers and relative structural changes studied by fluorescence microscopy

Our study is to understand the mechanism of ceramide production, then depletion of cholesterol and relative structural changes in supported raft bilayers using fluorescence microscopy. We worked with 5 nm thick supported raft bilayers of various compositions prepared by vesicle fusion method. The supported raft bilayers were treated with sphingomyelinase to understand hydrolysis of sphingomyelin at 37 °C and higher temperatures. We wanted to explore if the hydrolysis of sphingomyelin would produce ceramide and/or any other molecules. We were interested in knowing, what would be the structural changes in the raft bilayer due to reorganization of lipid molecules upon production of ceramide and other molecules? After the hydrolysis reaction, we saw bright spots and black patches on entire raft bilayer sample after production and reorganization. These bright spots were reversible three dimensional caps with the existing bilayer, which were proven by Fluorescence Recovery after Photo-bleaching. This concludes that excess molecules are present in the resultant bilayer, the heterogeneity of bilayer structure has resulted due to production of two molecules from single sphingomyelin i.e., phosphorylcholine and ceramide which are formed by cleavage SM making the bilayer rich in molecules. The heterogeneity might have resulted because of phase separation of cholesterol from ceramide. Since, they do not coexist as ceramide displaces cholesterol.

Molecular insights into the effect of an apoptotic raft-like bilayer on the conformation and dynamics of calreticulin

Cell surface calreticulin (CRT) can mediate apoptotic cells removal by binding and activating LDL receptor-related protein (LRP1). Phosphatidylserine (PS) lipids in the inner leaflet of the cell membrane are externalized and become exposed in cholesterol (CHOL)-rich membrane raft-like microdomain during apoptosis and co-localized with cell surface CRT. How the apoptotic raft-like membrane microdomain affects the structure and dynamics of CRT, further affecting CRT binding with LRP1 to signal apoptotic-cell clearance, remains unknown. In this study, we investigate the interactions between CRT and raft-like bilayers with or without POPS lipids with molecular dynamics simulations. In addition, the effect of an apoptotic raft bilayer on the binding between CRT and thrombospondin-1 (TSP1), a ligand of CRT on the cell surface to signal focal adhesion disassembly, was also investigated. Results of single CRT interactions with raft-like bilayers show that PS lipids in apoptotic raft-like bilayer increased the interactions between CRT and lipid bilayer, which enhanced the conformational stability and increased dynamical motion of CRT. The microscopic and mesoscopic properties of apoptotic raft-like bilayer were altered by the binding of CRT with lipid bilayer. Results of CRT-TSP1 complex interactions with raft-like bilayers show that the binding free energy between TSP1 and CRT was reduced in apoptotic raft-like bilayer environment. This study provides molecular and structural insight into the effect of an apoptotic raft-like bilayer on the conformation and dynamics of CRT, which could enrich our understanding of CRT-mediated apoptotic-cell clearance and focal adhesion disassembly.

Energy of the interaction between membrane lipid domains calculated from splay and tilt deformations

Specific domains, called rafts, are formed in cell membranes. Similar lipid domains can be formed in model membranes as a result of phase separation with raft size may remaining small (∼10–100 nm) for a long time. The characteristic lifetime of a nanoraft ensemble strongly depends on the nature of mutual raft interactions. The interaction energy between the boundaries of two rafts has been calculated under the assumption that the thickness of the raft bilayer is greater than that of the surrounding membrane, and elastic deformations appear in order to smooth the thickness mismatch at the boundary. When rafts approach each other, deformations from their boundaries overlap, making interaction energy profile sophisticated. It has been shown that raft merger occurs in two stages: rafts first merge in one monolayer of the lipid bilayer and then in another monolayer. Each merger stage requires overcoming of an energy barrier of about 0.08–0.12 kBT per 1 nm of boundary length. These results allow us to explain the stability of the ensemble of finite sized rafts.

Molecular simulation of rapid translocation of cholesterol, diacylglycerol, and ceramide in model raft and nonraft membranes

The translocation of lipids across membranes (flip-flop) is an important biological process. Slow exchange on a physiological timescale allows the creation of asymmetric distributions of lipids across cellular membranes. The location of lipids and their rate of exchange have important biological consequences, especially for lipids involved in cellular signaling. We investigated the translocation of cholesterol, ceramide, and diacylglycerol in two model bilayers using molecular dynamics simulations. We estimate half times for flip-flop for cholesterol, diacylglycerol, and ceramide of 20 μs, 30 μs, and 10 ms in a POPC bilayer, compared with approximately 30 min, 30 ms, and 30 s in a model raft bilayer (1:1:1 PSM, POPC, and cholesterol). Cholesterol has a large (54 kJ/mol) free energy of exchange between the POPC and raft bilayer, and therefore, it strongly prefers the more ordered and rigid raft bilayer over the more liquid POPC bilayer. Ceramide and diacylglycerol have relatively small free energies of exchange, suggesting nearly equal preference for both bilayers. This unexpected result may have implications for ceramide and diacylglycerol signaling and membrane localization.

Molecular Dynamics Simulations of Ceramide Flip-Flop and Desorption in Lipid Rafts

Ceramides are important signaling lipids, involved in processes such as apoptosis and cell differentiation. Upon stimulus acid sphingomyelinase hydrolyses sphingomyelin into ceramide on the extracellular leaflet. How ceramide generated on the extracellular leaflet induces an intracellular response remains unknown. Ceramide has been shown to associate and stabilize lipid rafts, which could create a signaling platform. We have undertaken molecular dynamics computer simulations (MD) of ceramide in both putative raft and non-raft bilayers. Using umbrella sampling we determined free energy profiles for moving ceramide and cholesterol from water to the center of raft and non-raft bilayers. The free energy barrier for ceramide flip-flop is 49 kJ/mol and 62 kJ/mol in the non-raft and raft bilayers. From these barriers, we estimate the rate of ceramide flip-flop is 0.3 s-1 and 0.003 s-1 in the non-raft and raft bilayers. The free energy for desorption can be equated to the excess chemical potential of ceramide in the bilayer compared to water. By comparing the chemical potentials, we can infer the relative affinity of ceramide and cholesterol for the raft and non-raft bilayers. Cholesterol has a large affinity for the raft bilayer compared to the non-raft bilayer, while ceramide has only a slight preference for the raft bilayer. These results provide a thermodynamic molecular-level description of the interactions of ceramide with lipid rafts, and the rate of translocation.

Role of GAP-43 in Sequestering Phosphatidylinositol 4,5-Bisphosphate to Raft Bilayers

The lipid phosphatidylinositol 4,5-bisphosphate (PIP 2) is critical for a number of physiological functions, and its presence in membrane microdomains (rafts) appears to be important for several of these spatially localized events. However, lipids like PIP 2 that contain polyunsaturated hydrocarbon chains are usually excluded from rafts, which are enriched in phospholipids (such as sphingomyelin) containing saturated or monounsaturated chains. Here we tested a mechanism by which multivalent PIP 2 molecules could be transferred into rafts through electrostatic interactions with polybasic cytoplasmic proteins, such as GAP-43, which bind to rafts via their acylated N-termini. We analyzed the interactions between lipid membranes containing raft microdomains and a peptide (GAP-43P) containing the linked N-terminus and the basic effector domain of GAP-43. In the absence or presence of nonacylated GAP-43P, PIP 2 was found primarily in detergent-soluble membranes thought to correspond to nonraft microdomains. However, when GAP-43P was acylated by palmitoyl coenzyme A, both the peptide and PIP 2 were greatly enriched in detergent-resistant membranes that correspond to rafts; acylation of GAP-43P changed the free energy of transfer of PIP 2 from detergent-soluble membranes to detergent-resistant membranes by −1.3 kcal/mol. Confocal microscopy of intact giant unilamellar vesicles verified that in the absence of GAP-43P PIP 2 was in nonraft microdomains, whereas acylated GAP-43P laterally sequestered PIP 2 into rafts. These data indicate that sequestration of PIP 2 to raft microdomains could involve interactions with acylated basic proteins such as GAP-43.

Assessing the Nature of Lipid Raft Membranes

The paradigm of biological membranes has recently gone through a major update. Instead of being fluid and homogeneous, recent studies suggest that membranes are characterized by transient domains with varying fluidity. In particular, a number of experimental studies have revealed the existence of highly ordered lateral domains rich in sphingomyelin and cholesterol (CHOL). These domains, called functional lipid rafts, have been suggested to take part in a variety of dynamic cellular processes such as membrane trafficking, signal transduction, and regulation of the activity of membrane proteins. However, despite the proposed importance of these domains, their properties, and even the precise nature of the lipid phases, have remained open issues mainly because the associated short time and length scales have posed a major challenge to experiments. In this work, we employ extensive atom-scale simulations to elucidate the properties of ternary raft mixtures with CHOL, palmitoylsphingomyelin (PSM), and palmitoyloleoylphosphatidylcholine. We simulate two bilayers of 1,024 lipids for 100 ns in the liquid-ordered phase and one system of the same size in the liquid-disordered phase. The studies provide evidence that the presence of PSM and CHOL in raft-like membranes leads to strongly packed and rigid bilayers. We also find that the simulated raft bilayers are characterized by nanoscale lateral heterogeneity, though the slow lateral diffusion renders the interpretation of the observed lateral heterogeneity more difficult. The findings reveal aspects of the role of favored (specific) lipid–lipid interactions within rafts and clarify the prominent role of CHOL in altering the properties of the membrane locally in its neighborhood. Also, we show that the presence of PSM and CHOL in rafts leads to intriguing lateral pressure profiles that are distinctly different from corresponding profiles in nonraft-like membranes. The results propose that the functioning of certain classes of membrane proteins is regulated by changes in the lateral pressure profile, which can be altered by a change in lipid content.

Transbilayer Peptide Sorting between Raft and Nonraft Bilayers: Comparisons of Detergent Extraction and Confocal Microscopy

Membrane microdomains (“rafts”) that sequester specific proteins and lipids are often characterized by their resistance to detergent extraction. Because rafts are enriched in sphingomyelin and cholesterol, raft bilayers are thicker and have larger area compressibility moduli than nonraft bilayers. It has been postulated that rafts concentrate proteins with long transmembrane domains (TMDs) because of “hydrophobic matching” between the TMDs and the thick raft bilayers. However, previous detergent extraction experiments with bilayers containing raft and nonraft domains have shown that the peptides P-23 and P-29, designed to have single TMDs matching the hydrocarbon thicknesses of detergent soluble membranes and detergent resistant membranes, respectively, are both localized to detergent soluble membranes. Those results imply that both peptides are preferentially located in nonraft domains. However, because the detergent solubilizes part of the bilayer, it has been unclear whether or not detergent extraction experiments provide an accurate indication of the location of peptides in intact bilayers. Here we use confocal microscopy to examine the distribution of these same peptides in intact bilayers containing both raft and nonraft domains. At 20°C and 37°C, P-23 and P-29 were both primarily localized in fluorescently labeled nonraft domains. These confocal results validate the previous detergent extraction experiments and demonstrate the importance of bilayer cohesive properties, compared to hydrophobic mismatch, in the sorting of these peptides that contain a single TMD.

The inner side of T cell lipid rafts

A key question in understanding the functional role of lipid rafts is whether lipid microdomains at the plasma membrane outer leaflet are coupled to lipid microdomains at the inner leaflet. By using a cyan-fluorescent protein (CFP) targeted to inner plasma membrane rafts of Jurkat T cells, we found that raft domains at the outer and inner leaflets are physically coupled and that this coupling requires cholesterol. Interestingly, TCR/CD3 cross-linking induces co-capping of the raft bilayer independently of cholesterol or signaling events, indicating that cholesterol-extracting drugs are unable to destroy TCR–lipid rafts interaction.

Sorting of Lipids and Transmembrane Peptides Between Detergent-Soluble Bilayers and Detergent-Resistant Rafts

Specific proteins and lipids sequester to regions of cell membranes called rafts. Due to their high content of sphingomyelin (SM) and cholesterol, raft bilayers are thicker than nonraft bilayers and, at least at 4°C, are resistant to Triton X-100 extraction. It has been postulated that rafts concentrate proteins with long transbilayer domains because of “hydrophobic matching” between the transbilayer domain and the thick bilayer hydrocarbon region. However, because the area compressibility and bending moduli of SM:cholesterol bilayers are larger than that of nonraft bilayers, there should be an energy cost to partition proteins or peptides into rafts. To determine the effects on peptide sorting of raft thickness and mechanical properties, we incorporated two transbilayer peptides (P-23, P-29) into bilayers composed of SM, dioleoylphosphatidylcholine, and cholesterol, separated detergent-soluble membranes (DSMs) from detergent-resistant membranes (DRMs), and measured their peptide and lipid compositions. P-23 and P-29 were designed to have transbilayer domains that matched the hydrocarbon thicknesses of DSMs and DRMs, respectively. At both 4°C and 37°C DSMs were enriched in dioleoylphosphatidylcholine and DRMs were enriched in SM and cholesterol. At both temperatures both P-23 and P-29 preferentially localized to DSMs, demonstrating the importance of bilayer mechanical properties relative to hydrophobic mismatch. However, at 37°C significantly more P-29 than P-23 was located in DRMs, implying that hydrophobic matching played a role in peptide sorting at physiological temperature. These experiments demonstrate that the sorting of peptides as measured by detergent extraction is temperature-dependent and both bilayer mechanical properties and hydrophobic matching impact peptide distribution between DSMs and DRMs.