Liquid-ordered Membrane Research Articles

Interaction Forces between Lipid Rafts.

Cellular membranes containing sphingolipids and cholesterol have been shown to self-organize into lipid rafts-specialized domains that host integral membrane proteins and modulate the bioactivity of cells. In this work, force-distance profiles between raft membranes in the liquid-ordered phase consisting of singly unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a complex mixture of brain sphingomyelin (BSM), and cholesterol were measured using the surface force apparatus (SFA). Two distinct force profiles were detected corresponding to uniform raft membranes and raft membranes with a higher level of topological membrane defects (heterogeneous) as corroborated by atomic force microscopy (AFM) scans. In all cases a weak, long-range electrostatic repulsion was observed with some variation in the surface charge density. The variation in electrostatic repulsion was attributed to charged lipid species primarily from the constituent lipids in the BSM mixture. The adhesion between the uniform raft membranes was comparable to our previous work with pure component, liquid-ordered POPC-DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine)-cholesterol membranes. Raft membranes with more topological defects adhered more strongly owing to hydrophobic attraction between exposed acyl chains. Even though the rafts were in the liquid-ordered phase and membrane defects were present in the contact region, the raft membranes were stable, and no structural rearrangement was observed throughout the measurements. Our findings demonstrate that liquid-ordered membranes are stable to mechanical loading and not particularly sensitive to compositional variation.

Effect of local anesthetics on the organization and dynamics in membranes of varying phase: A fluorescence approach

The molecular mechanism underlying the action of local anesthetics remains elusive. Phenylethanol (PEtOH) is an ingredient of essential oils with a rose-like odor and has been used as a local anesthetic. In this work, we explored the effect of PEtOH on organization and dynamics in membranes representing various biologically relevant phases using differentially localized fluorescent membrane probes, DPH and TMA-DPH. We show here that PEtOH induces disorder in membranes of all phases (gel/fluid/liquid-ordered). However, the extent of membrane disorder varies in a phase-specific manner. Maximum membrane disordering was observed in gel phase, followed by liquid-ordered membranes. The disordering was minimal in fluid phase membranes. Interestingly, our results show that the disordering effect of PEtOH in gel phase is sufficiently large to induce phase change at higher PEtOH concentrations. Our results are relevant in the context of natural membranes and could be useful in understanding the role of anesthetics in membrane protein function.

Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking.

Lipid rafts are membrane nanodomains that facilitate important cell functions. Despite recent advances in identifying the biological significance of rafts, nature and regulation mechanism of rafts are largely unknown due to the difficulty of resolving dynamic molecular interaction of rafts at the nanoscale. Here, we investigate organization and single-molecule dynamics of rafts by monitoring lateral diffusion of single molecules in raft-containing reconstituted membranes supported on mica substrates. Using high-speed interferometric scattering (iSCAT) optical microscopy and small gold nanoparticles as labels, motion of single lipids is recorded via single-particle tracking (SPT) with nanometer spatial precision and microsecond temporal resolution. Processes of single molecules partitioning into and escaping from the raft-mimetic liquid-ordered (Lo) domains are directly visualized in a continuous manner with unprecedented clarity. Importantly, we observe subdiffusion of saturated lipids in the Lo domain in microsecond timescale, indicating the nanoscopic heterogeneous molecular arrangement of the Lo domain. Further analysis of the diffusion trajectory shows the presence of nano-subdomains of the Lo phase, as small as 10 nm, which transiently trap the lipids. Our results provide the first experimental evidence of non-uniform molecular organization of the Lo phase, giving a new view of how rafts recruit and confine molecules in cell membranes.

Nano-Substructures of raft-Mimetic Liquid-Ordered Membrane Domains Revealed by High-Speed Single-Particle Tracking

Biological membrane domains of distinct molecular compositions and organizations facilitate many important membrane functions. Lipid rafts are specialized membrane domains of liquid-ordered (Lo) phase that are found to be essential for various membrane processes. Despite of recent advances in identifying the biological significance of rafts, the dynamic nature and regulation mechanism of rafts are largely unknown due to the lack of technology to resolve the transient molecular interaction with rafts at the nanoscale. In this work, we investigate how rafts recruit and trap molecules by monitoring lateral diffusion of single lipids in raft-containing reconstituted membranes at ultrahigh spatiotemporal resolution. Using high-speed interferometric scattering (iSCAT) optical microscopy and small gold nanoparticles as labels, the motion of single lipids in the membrane is recorded via single-particle tracking (SPT) with 3 nm spatial precision and 20 μs temporal resolution. The processes of single molecules partitioning into and escaping from the raft-mimetic Lo domains are directly visualized in a continuous manner with unprecedented clarity. Surprisingly, we observe anomalous subdiffusion of saturated lipids in the Lo domain in microsecond timescale, indicating the nanoscopic heterogeneous molecular arrangement of the Lo domain. Further analysis of the diffusion trajectory shows the presence of nano-subdomains of the Lo phase, as small as 10 nm, that transiently trap the saturated lipids. Our data provide the first experimental evidence of non-uniform molecular organization of the Lo phase and show its critical role in determining molecular dynamics in the Lo phase. Our results also imply that the raft-associated molecules could be trapped between nano-subdomains of densely packed saturated lipids, surrounded by high density of cholesterol. The nanoscopic membrane environment created by the nano-subdomains of rafts is thus unique, which illustrates the essentials of lipid rafts of Lo phase in cell membrane.

Modeling the effects of cyclodextrin on intracellular membrane vesicles from Cos-7 cells prepared by sonication and carbonate treatment.

Cholesterol has important functions in the organization of membrane structure and this may be mediated via the formation of cholesterol-rich, liquid-ordered membrane microdomains often referred to as lipid rafts. Methyl-beta-cyclodextrin (cyclodextrin) is commonly used in cell biology studies to extract cholesterol and therefore disrupt lipid rafts. However, in this study we reassessed this experimental strategy and investigated the effects of cyclodextrin on the physical properties of sonicated and carbonate-treated intracellular membrane vesicles isolated from Cos-7 fibroblasts. We treated these membranes, which mainly originate from the trans-Golgi network and endosomes, with cyclodextrin and measured the effects on their equilibrium buoyant density, protein content, represented by the palmitoylated protein phosphatidylinositol 4-kinase type IIα, and cholesterol. Despite the reduction in mass stemming from cholesterol removal, the vesicles became denser, indicating a possible large volumetric decrease, and this was confirmed by measurements of hydrodynamic vesicle size. Subsequent mathematical analyses demonstrated that only half of this change in membrane size was attributable to cholesterol loss. Hence, the non-selective desorption properties of cyclodextrin are also involved in membrane size and density changes. These findings may have implications for preceding studies that interpreted cyclodextrin-induced changes to membrane biochemistry in the context of lipid raft disruption without taking into account our finding that cyclodextrin treatment also reduces membrane size.

Multiple locations of peptides in the hydrocarbon core of gel-phase membranes revealed by peptide (13)C to lipid (2)H rotational-echo double-resonance solid-state nuclear magnetic resonance.

Membrane locations of peptides and proteins are often critical to their functions. Solid-state rotational-echo double-resonance (REDOR) nuclear magnetic resonance is applied to probe the locations of two peptides via peptide 13CO to lipid 2H distance measurements. The peptides are KALP, an α-helical membrane-spanning peptide, and HFP, the β-sheet N-terminal fusion peptide of the HIV gp41 fusion protein that plays an important role in HIV–host cell membrane fusion. Both peptides are shown to have at least two distinct locations within the hydrocarbon core of gel-phase membranes. The multiple locations are attributed to snorkeling of lysine side chains for KALP and to the distribution of antiparallel β-sheet registries for HFP. The relative population of each location is also quantitated. To the best of our knowledge, this is the first clear experimental support of multiple peptide locations within the membrane hydrocarbon core. These data are for gel-phase membranes, but the approach should work for liquid-ordered membranes containing cholesterol and may be applicable to liquid-disordered membranes with appropriate additional analysis to take into account protein and lipid motion. This paper also describes the methodological development of 13CO–2H REDOR using the lyophilized I4 peptide that is α-helical and 13CO-labeled at A9 and 2Hα-labeled at A8. The I4 spins are well-approximated as an ensemble of isolated 13CO–2H spin pairs each separated by 5.0 Å with a 37 Hz dipolar coupling. A pulse sequence with rectangular 100 kHz 2H π pulses results in rapid and extensive buildup of REDOR (ΔS/S0) with a dephasing time (τ). The buildup is well-fit by a simple exponential function with a rate of 24 Hz and an extent close to 1. These parameter values reflect nonradiative transitions between the 2H spin states during the dephasing period. Each spin pair spends approximately two-thirds of its time in the 13CO–2H (m = ±1) states and approximately one-third of its time in the 13CO–2H (m = 0) state and contributes to the ΔS/S0 buildup during the former but not the latter time segments.

Interaction of NBD-labelled fatty amines with liquid-ordered membranes: a combined molecular dynamics simulation and fluorescence spectroscopy study.

A complete homologous series of fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl-(NBD) labelled fatty amines of varying alkyl chain lengths, NBD-Cn, inserted in 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine (POPC) or N-palmitoyl sphingomyelin (SpM) bilayers, with 50 mol% and 40 mol% cholesterol (Chol), respectively, was studied using atomistic molecular dynamics simulations. For all amphiphiles in both bilayers, the NBD fluorophore locates at the interface, in a more external position than that previously observed for pure POPC bilayers. This shallower location of the NBD group agrees with the lower fluorescent quantum yield, shorter fluorescence lifetime, and higher ionisation constants (smaller pKa) determined experimentally. The more external location is also consistent with the changes measured in steady-state fluorescence anisotropy from POPC to POPC/Chol (1 : 1) vesicles. Accordingly, the equilibrium location of the NBD group within the various bilayers is mainly dictated by bilayer compositions, and is mostly unaffected by the length of the attached alkyl chain. Similarly to the behaviour observed in POPC bilayers, the longer-chained NBD-Cn amphiphiles show significant mass density near the mixed bilayers' midplanes, and the alkyl chains of the longer derivatives, mainly NBD-C16, penetrate the opposite bilayer leaflet to some extent. However, this effect is quantitatively less pronounced in these ordered bilayers than in POPC. Similarly to POPC bilayers, the effects of these amphiphiles on the structure and dynamics of the host lipid were found to be relatively mild, in comparison with acyl-chain phospholipid analogues.

The myelin proteolipid plasmolipin forms oligomers and induces liquid-ordered membranes in the Golgi complex.

Myelin comprises a compactly stacked massive surface area of protein-poor thick membrane that insulates axons to allow fast signal propagation. Increasing levels of the myelin protein plasmolipin (PLLP) were correlated with post-natal myelination; however, its function is unknown. Here, the intracellular localization and dynamics of PLLP were characterized in primary glial and cultured cells using fluorescently labeled PLLP and antibodies against PLLP. PLLP localized to and recycled between the plasma membrane and the Golgi complex. In the Golgi complex, PLLP forms oligomers based on fluorescence resonance energy transfer (FRET) analyses. PLLP oligomers blocked Golgi to plasma membrane transport of the secretory protein vesicular stomatitis virus G protein (VSVG), but not of a VSVG mutant with an elongated transmembrane domain. Laurdan staining analysis showed that this block is associated with PLLP-induced proliferation of liquid-ordered membranes. These findings show the capacity of PLLP to assemble potential myelin membrane precursor domains at the Golgi complex through its oligomerization and ability to attract liquid-ordered lipids. These data support a model in which PLLP functions in myelin biogenesis through organization of myelin liquid-ordered membranes in the Golgi complex.

Lipid rafts as major platforms for signaling regulation in cancer

Cell signaling does not apparently occur randomly over the cell surface, but it seems to be integrated very often into cholesterol-rich membrane domains, termed lipid rafts. Membrane lipid rafts are highly ordered membrane domains that are enriched in cholesterol, sphingolipids and gangliosides, and behave as major modulators of membrane geometry, lateral movement of molecules, traffic and signal transduction. Because the lipid and protein composition of membrane rafts differs from that of the surrounding membrane, they provide an additional level of compartmentalization, serving as sorting platforms and hubs for signal transduction proteins. A wide number of signal transduction processes related to cell adhesion, migration, as well as to cell survival and proliferation, which play major roles in cancer development and progression, are dependent on lipid rafts. Despite lipid rafts harbor mainly critical survival signaling pathways, including insulin-like growth factor I (IGF-I)/phosphatidylinositol 3-kinase (PI3K)/Akt signaling, recent evidence suggests that these membrane domains can also house death receptor-mediated apoptotic signaling. Recruitment of this death receptor signaling pathway in membrane rafts can be pharmacologically modulated, thus opening up the possibility to regulate cell demise with a therapeutic use. The synthetic ether phospholipid edelfosine shows a high affinity for cholesterol and accumulates in lipid rafts in a number of malignant hematological cells, leading to an efficient invitro and invivo antitumor activity by inducing translocation of death receptors and downstream signaling molecules to these membrane domains. Additional antitumor drugs have also been shown to act, at least in part, by recruiting death receptors in lipid rafts. The partition of death receptors together with downstream apoptotic signaling molecules in membrane rafts has led us to postulate the concept of a special liquid-ordered membrane platform coined as "cluster of apoptotic signaling molecule-enriched rafts" (CASMER), referring to raft platforms enriched in apoptotic molecules. CASMERs act as scaffolds for apoptosis signaling compartmentalization, facilitating and stabilizing protein-protein interactions by local assembly of cross-interacting molecules, which leads to apoptosis amplification and a decrease in apoptotic signal threshold. Edelfosine also displaced survival PI3K/Akt signaling from lipid rafts, leading to Akt inhibition, in mantle cell lymphoma cells. Thus, membrane rafts could act as scaffold structures where segregation of pro- from anti-apoptotic molecules could take place. In this review, we summarize our view of how reorganization of the protein composition of lipid raft membrane domains regulates cell death and therefore it might be envisaged as a novel target in the treatment of cancer.

Improvement of HIV fusion inhibitor C34 efficacy by membrane anchoring and enhanced exposure

The aim of the present work was to evaluate the interaction of two new HIV fusion inhibitors {HIVP3 [C34-polyethylene glycol (PEG)₄-cholesterol] and HIVP4 [(C34-PEG₄)₂-cholesterol]} with membrane model systems and human blood cells in order to clarify where and how the fusion inhibitors locate, allowing us to understand their mechanism of action at the molecular level, and which strategies may be followed to increase efficacy. Lipid vesicles with defined compositions were used for peptide partition and localization studies, based on the intrinsic fluorescence of HIVP3 and HIVP4. Lipid monolayers were employed in surface pressure studies. Finally, human erythrocytes and peripheral blood mononuclear cells (PBMCs) isolated from blood samples were used in dipole potential assays. Membrane partition, dipole potential and surface pressure assays indicate that the new fusion inhibitors interact preferentially with cholesterol-rich liquid-ordered membranes, mimicking biological membrane microdomains known as lipid rafts. HIVP3 and HIVP4 are able to interact with human erythrocytes and PBMCs to a similar degree as a previously described simpler drug with monomeric C34 and lacking the PEG spacer, C34-cholesterol. However, the pocket-binding domain (PBD) of both HIVP3 and HIVP4 is more exposed to the aqueous environment than in C34-cholesterol. The present data allow us to conclude that more efficient blocking of HIV entry results from the synergism between the membranotropic behaviour and the enhanced exposure of the PBD.

Regulation of Rac translocation and activation by membrane domains and their boundaries

The activation of Rac1 and related Rho GTPases involves dissociation from Rho GDP-dissociation inhibitor proteins and translocation to membranes, where they bind effectors. Previous studies have suggested that the binding of Rac1 to membranes requires, and colocalizes with, cholesterol-rich liquid-ordered (lo) membrane domains (lipid rafts). Here, we have developed a fluorescence resonance energy transfer (FRET) assay that robustly detects Rac1 membrane targeting in living cells. Surprisingly, FRET with acceptor constructs that were targeted to either raft or non-raft areas indicated that Rac1 was present in both regions. Functional studies showed that Rac1 localization to non-raft regions decreased GTP loading as a result of inactivation by GTPase-activating proteins. In vitro, Rac1 translocation to supported lipid bilayers also required lo domains, yet Rac1 was concentrated in the liquid-disordered (ld) phase. Single-molecule analysis demonstrated that translocation occurred preferentially at lo-ld boundaries. These results, therefore, suggest that Rac1 translocates to the membrane at domain boundaries, then diffuses into raft and non-raft domains, which controls interactions. These findings resolve discrepancies in our understanding of Rac biology and identify novel mechanisms by which lipid rafts modulate Rho GTPase signaling.

Steric Pressure between Membrane-Bound Proteins Opposes Lipid Phase Separation

Cellular membranes are densely crowded with a diverse population of integral and membrane-associated proteins. In this complex environment, lipid rafts, which are phase-separated membrane domains enriched in cholesterol and saturated lipids, are thought to organize the membrane surface. Specifically, rafts may help to concentrate proteins and lipids locally, enabling cellular processes such as assembly of caveolae, budding of enveloped viruses, and sorting of lipids and proteins in the Golgi. However, the ability of rafts to concentrate protein species has not been quantified experimentally. Here we show that when membrane-bound proteins become densely crowded within liquid-ordered membrane regions, steric pressure arising from collisions between proteins can destabilize lipid phase separations, resulting in a homogeneous distribution of proteins and lipids over the membrane surface. Using a reconstituted system of lipid vesicles and recombinant proteins, we demonstrate that protein-protein steric pressure creates an energetic barrier to the stability of phase-separated membrane domains that increases in significance as the molecular weight of the proteins increases. Comparison with a simple analytical model reveals that domains are destabilized when the steric pressure exceeds the approximate enthalpy of membrane mixing. These results suggest that a subtle balance of free energies governs the stability of phase-separated cellular membranes, providing a new perspective on the role of lipid rafts as concentrators of membrane proteins.

Quantifying the Effect of BCR Clustering on Plasma Membrane Organization

The B cell antigen receptor (BCR) is an integral part of the adaptive immune system that communicates binding of antigen in the extracellular environment through the plasma membrane. Antigen binding to the BCR results in phosphorylation of intracellular tyrosine activating motifs (ITAMs), which subsequently bind to and activate numerous proteins involved in BCR regulation. Interestingly, many of the proteins regulating the early stages of the BCR signaling pathway are linked to the inner leaflet by saturated lipid anchors which tend to be associated with liquid-ordered membrane phase in model membranes. Also, the BCR becomes transiently detergent resistant following antigen-induced BCR clustering, suggesting that BCR clusters become coupled to membrane order following stimulation. In this work, we aim to characterize how BCR clustering could reorganize plasma membrane lipids by quantifying co-localization of BCR with fluorescent markers of liquid-ordered and liquid-disordered phases. We utilize two-color super-resolution fluorescence localization microscopy (STORM and PALM) in live and chemically fixed CH27 B cells to simultaneously image BCR and membrane anchored proteins, and we quantify their co-clustering using correlation functions. Our results from chemically fixed cells show that proteins anchored to the plasma membrane inner leaflet through saturated acyl-chain lipid modifications exhibit increased co-localization with BCR upon antigen stimulation, whereas those without lipid modifications or those anchored through branched acyl-chain modifications are not significantly co-localized with BCR before or after stimulation. These results are contributing to our long term goal of elucidating the role of lipid mediated interactions in the regulation of BCR signaling.

Detergent-resistant Membranes-association of the Interleukin-6 Signal Transducer gp130 and its Mutant gp130LL/AA Differs with the Methods of Isolation

The transmembrane protein gp130 is the common signal transducer for the interleukin-6 (IL-6) type cytokines. It has been shown that cytoplasmic dileucine internalization motif also directs gp130 to the basolateral plasma membrane of polarized MDCK cells and protein partially localizes to detergentresistant membranes (DRMs)/lipid rafts. Here we have studied whether the same dileucine motif is also raft association signal, similarly to sorting signals. Using two different lipid raft detergent-extraction protocols, we have found that wild type gp130, truncated gp130 lacking the cytoplasmic domain and gp130 mutant in which the di-leucine motif has been exchanged by two alanines have equal affinity for the liquid-ordered membrane domain independently of the methods of isolation. We demonstrate difference in cofractionation, fully or partially with lipid rafts of gp130wt and mutants depending on approach. Despite the differences, dileucine motif, which is both internalization and sorting one, is not a raft targeting signal.

Molecular view on protein sorting into liquid-ordered membrane domains mediated by gangliosides and lipid anchors

We present results from coarse grain molecular dynamics simulations of mixed model membranes consisting of saturated and unsaturated lipids together with cholesterol, in which lipid-anchored membrane proteins are embedded. The membrane proteins studied are the peripherally bound H-Ras, N-Ras, and Hedgehog, and the transmembrane peptides WALP and LAT. We provide a molecular view on how the presence and nature of these lipid anchors affects partitioning of the proteins between liquid-ordered and liquid-disordered domains. In addition, we probed the role of the ganglioside lipid GM1 on the protein sorting, showing formation of GM1-protein nano-domains that act as shuttles between the differently ordered membrane regions.

Organization and Dynamics of Fas Transmembrane Domain in Raft Membranes and Modulation by Ceramide

To comprehend the molecular processes that lead to the Fas death receptor clustering in lipid rafts, a 21-mer peptide corresponding to its single transmembrane domain (TMD) was reconstituted into mammalian raft model membranes composed of an unsaturated glycerophospholipid, sphingomyelin, and cholesterol. The peptide membrane lateral organization and dynamics, and its influence on membrane properties, were studied by steady-state and time-resolved fluorescence techniques and by attenuated total reflection Fourier transformed infrared spectroscopy. Our results show that Fas TMD is preferentially localized in liquid-disordered membrane regions and undergoes a strong reorganization as the membrane composition is changed toward the liquid-ordered phase. This results from the strong hydrophobic mismatch between the length of the peptide hydrophobic stretch and the hydrophobic thickness of liquid-ordered membranes. The stability of nonclustered Fas TMD in liquid-disordered domains suggests that its sequence may have a protective function against nonligand-induced Fas clustering in lipid rafts. It has been reported that ceramide induces Fas oligomerization in lipid rafts. Here, it is shown that neither Fas TMD membrane organization nor its conformation is affected by ceramide. These results are discussed within the framework of Fas membrane signaling events.

Dihydrosphingomyelin Impairs HIV-1 infection by Rigidifying Liquid-Ordered Membrane Domains

The lateral organization of lipids in cell membranes is thought to regulate numerous cell processes. Most studies focus on the coexistence of two fluid phases, the liquid crystalline (ld) and the liquid-ordered (lo); the putative presence of gel domains (so) is not usually taken into account. We show that in phospholipid:sphingolipid:cholesterol mixtures, in which sphingomyelin (SM) promoted fluid lo domains, dihydrosphingomyelin (DHSM) tended to form rigid domains. Genetic and pharmacological blockade of the dihydroceramide desaturase (Des1), which replaced SM with DHSM in cultured cells, inhibited cell infection by replication-competent and deficient HIV-1. Increased DHSM levels gave rise to more rigid membranes, resistant to the insertion of the gp41 fusion peptide, thus inhibiting viral-cell membrane fusion. These results clarify the function of dihydrosphingolipids in biological membranes and identify Des1 as a potential target in HIV-1 infection.

Dihydrosphingomyelin Impairs HIV-1 Infection by Rigidifying Liquid-Ordered Membrane Domains

The lateral organization of lipids in cell membranes is thought to regulate numerous cell processes. Most studies focus on the coexistence of two fluid phases, the liquid crystalline (l(d)) and the liquid-ordered (l(o)); the putative presence of gel domains (s(o)) is not usually taken into account. We show that in phospholipid:sphingolipid:cholesterol mixtures, in which sphingomyelin (SM) promoted fluid l(o) domains, dihydrosphingomyelin (DHSM) tended to form rigid domains. Genetic and pharmacological blockade of the dihydroceramide desaturase (Des1), which replaced SM with DHSM in cultured cells, inhibited cell infection by replication-competent and -deficient HIV-1. Increased DHSM levels gave rise to more rigid membranes, resistant to the insertion of the gp41 fusion peptide, thus inhibiting viral-cell membrane fusion. These results clarify the function of dihydrosphingolipids in biological membranes and identify Des1 as a potential target in HIV-1 infection.

Do local anesthetics interact preferentially with membrane lipid rafts? Comparative interactivities with raft-like membranes

Membranous lipid bilayers have been reconsidered as the site of action of local anesthetics (LAs). Recent understanding of biomembranes indicates the existence of lipid raft microdomains enriched in cholesterol and sphingolipids as potential platforms for channels and receptors. Based on the hypothesis that LAs may interact preferentially with lipid rafts over non-raft membranes, we compared their effects on raft model membranes and cardiolipin-containing biomimetic membranes. Liposomes were prepared with phospholipids, sphingomyelin, cerebroside, and cholesterol to have compositions corresponding to lipid rafts and cardiomyocyte mitochondrial membranes. After reacting LAs (50-200 microM) with the membrane preparations, their interactivities were determined by measuring fluorescence polarization with 1,6-diphenyl-1,3,5-hexatriene. Although bupivacaine and lidocaine acted on different raft-like liquid-ordered membranes to reduce polarization values, their effects on biomimetic less ordered membranes were much greater. LAs interacted with biomimetic membranes with the potency being R(+)-bupivacaine > racemic bupivacaine > S(-)-bupivacaine > ropivacaine > lidocaine > prilocaine, which is consistent with the rank order of pharmacotoxicological potency. However, raft model membranes showed neither structure-dependence nor stereoselectivity. The relevance of membrane lipid rafts to LAs is questionable at least in their effects on raft-like liquid-ordered membranes.

Differentiating Lipid Phase Domains in Cells using Fluorescence Lifetime of DiI

Dynamic heterogeneous distribution of lipid phases is thought to be an important means by which lipids modulate cellular biology. However, information about these domains is not accessible using conventional optical microscopy because their size (10-100nm) is well below the diffraction limit. Fluorescence lifetime (FL) of variable chain length di-alkyl carbocyanine dyes (DiIs) has been proposed to reflect lipid phase, but verification that fluorescence readouts reflect lipid phase in cells is lacking. Thus, we used time-correlated single photon counting techniques to test the fidelity with which photophysical properties of DiI-C12 and DiI-C18 report lipid order in cells. DiI FL increases with increased solution viscosity (1 to 70 cP) were independent of acyl chain length, demonstrating that lifetime reports chromophore headgroup local viscosity. Sensitivity of FL of DiI to membrane order was evaluated in giant unilamellar vesicles (GUVs) composed of DOPC (liquid-disordered), DOPC:Chol (liquid ordered), DPPC:Chol (liquid ordered), and DPPC (gel phase). FL of DiI increased significantly with increase in membrane order, and correlated well with a decrease in diffusion coefficient. Phase partitioning of DiI-C12 and DiI-C18 was characterized in phase-coexistent GUVs composed of DOPC:DPPC:Chol (2:2:1). Short chain DiI-C12 and long chain DiI-C18 partitioned to liquid-disordered and liquid-ordered membrane phases, respectively. FL of DiI-C18 stained cells (1.47 ± 0.49 ns) was higher compared to DiI-C12 stained cells (1.26 ± 0.12 ns), indicating that DiI-C18 and DiI-C12 partitioned into liquid-ordered and liquid-disordered phases respectively. In conclusion, FL of DiI is a sensitive indicator of membrane fluidity and different chain length DiI's partition to different membrane phases both in model and cell plasma membranes.