Temperature dependence of diffusion in model and live cell membranes characterized by imaging fluorescence correlation spectroscopy

The organization of the plasma membrane is regulated by the dynamic equilibrium between the liquid ordered (L_o) and liquid disordered (L_d) phases. The abundance of the L_o phase is assumed to be a consequence of the interaction between cholesterol and the other lipids, which are otherwise in either the L_d or gel (S_o) phase. The characteristic lipid packing in these phases results in significant differences in their respective lateral dynamics. In this study, imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) is applied to monitor the diffusion within supported lipid bilayers (SLBs) as functions of temperature and composition. We show that the temperature dependence of membrane lateral diffusion, which is parameterized by the Arrhenius activation energy (E_Arr), can resolve the sub-resolution phase behavior of lipid mixtures. The FCS diffusion law, a novel membrane heterogeneity ruler implemented in ITIR-FCS, is applied to show that the domains in the S_o–L_d phase are static and large while they are small and dynamic in the L_o–L_d phase. Diffusion measurements and the subsequent FCS diffusion law analyses at different temperatures show that the modulation in membrane dynamics at high temperature (313 K) is a cumulative effect of domain melting and rigidity relaxation. Finally, we extend these studies to the plasma membranes of commonly used neuroblastoma, HeLa and fibroblast cells. The temperature dependence of membrane dynamics for neuroblastoma cells is significantly different from that of HeLa or fibroblast cells as the different cell types exhibit a high level of compositional heterogeneity.     

Membrane Fluidity and Lipid Order in Ternary Giant Unilamellar Vesicles Using a New Bodipy-Cholesterol Derivative

Cholesterol-rich, liquid-ordered (L_o) domains are believed to be biologically relevant, and yet detailed knowledge about them, especially in live cells under physiological conditions, is elusive. Although these domains have been observed in model membranes, understanding cholesterol-lipid interactions at the molecular level, under controlled lipid mixing, remains a challenge. Further, although there are a number of fluorescent lipid analogs that partition into liquid-disordered (L_d) domains, the number of such analogs with a high affinity for biologically relevant L_o domains is limited. Here, we use a new Bodipy-labeled cholesterol (Bdp-Chol) derivative to investigate membrane fluidity, lipid order, and partitioning in various lipid phases in giant unilamellar vesicles (GUVs) as a model system. GUVs were prepared from mixtures of various molar fractions of dioleoylphosphatidylcholine, cholesterol, and egg sphingomyelin. The L_d phase domains were also labeled with 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI-C₁₂) for comparison. Two-photon fluorescence lifetime and anisotropy imaging of Bdp-Chol are sensitive to lipid phase domains in GUVs. The fluorescence lifetime of Bdp-Chol in liquid-disordered, single-phase GUVs is 5.50 ± 0.08 ns, compared with 4.1 ± 0.4 ns in the presence of DiI-C₁₂. The observed reduction of fluorescence lifetime is attributed to Förster resonance energy transfer between Bdp-Chol (a donor) and DiI-C₁₂ (an acceptor) with an estimated efficiency of 0.25 and donor-acceptor distance of 2.6 ± 0.2 nm. These results also indicate preferential partitioning (K_p = 1.88) of Bdp-Chol into the L_o phase. One-photon, time-resolved fluorescence anisotropy of Bdp-Chol decays as a triexponential in the lipid bilayer with an average rotational diffusion coefficient, lipid order parameter, and membrane fluidity that are sensitive to phase domains. The translational diffusion coefficient of Bdp-Chol, as measured using fluorescence correlation spectroscopy, is (7.4 ± 0.3) × 10⁻⁸ cm²/s and (5.0 ± 0.2) × 10⁻⁸ cm²/s in the L_d and L_o phases, respectively. Experimental translational/rotational diffusion coefficient ratios are compared with theoretical predictions using the hydrodynamic model (Saffman-Delbrück). The results suggest that Bdp-Chol is likely to form a complex with other lipid molecules during its macroscopic diffusion in GUV lipid bilayers at room temperature. Our integrated, multiscale results demonstrate the potential of this cholesterol analog for studying lipid-lipid interactions, lipid order, and membrane fluidity of biologically relevant L_o domains.     

Line tension at lipid phase boundaries regulates formation of membrane vesicles in living cells

Ternary lipid compositions in model membranes segregate into large-scale liquid-ordered (L_o) and liquid-disordered (L_d) phases. Here, we show μm-sized lipid domain separation leading to vesicle formation in unperturbed human HaCaT keratinocytes. Budding vesicles in the apical portion of the plasma membrane were predominantly labelled with L_d markers 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate and weakly stained by L_o marker fluorescein-labeled cholera toxin B subunit which labels ganglioside GM₁ enriched plasma membrane rafts. Cholesterol depletion with methyl-β-cyclodextrin enhanced DiI vesiculation, GM₁/DiI domain separation and was accompanied by a detachment of the subcortical cytoskeleton from the plasma membrane. Based on these observations we describe the energetic requirements for plasma membrane vesiculation. We propose that the decrease in total ‘L_o/L_d’ boundary line tension arising from the coalescence of smaller L_d-like domains makes it energetically favourable for L_d-like domains to bend from flat μm-sized surfaces to cap-like budding vesicles. Thus living cells may utilize membrane line tension energies as a control mechanism of exocytic events.     

Fluorescence probe partitioning between Lo/Ld phases in lipid membranes

Fluorescence microscopy imaging is an important technique for studying lipid membranes and is increasingly being used for examining lipid bilayer membranes, especially those showing macroscopic coexisting domains. Lipid phase coexistence is a phenomenon of potential biological significance. The identification of lipid membrane heterogeneity by fluorescence microscopy relies on membrane markers with well-defined partitioning behavior. While the partitioning of fluorophores between gel and liquid-disordered phases has been extensively characterized, the same is not true for coexisting liquid phases. We have used fluorescence microscopy imaging to examine a large variety of lipid membrane markers for their liquid phase partitioning in membranes with various lipid compositions. Most fluorescent lipid analogs are found to partition strongly into the liquid-disordered (L_d) phase. In contrast, some fluorescent polycyclic aromatic hydrocarbons with a flat ring system were found to partition equally, but others partition preferentially into liquid-ordered (L_o) phases. We have found these fluorescent markers effective for identification of coexisting macroscopic membrane phases in ternary lipid systems composed of phospholipids and cholesterol.     

Lo/Ld phase coexistence modulation induced by GM1

Lipid rafts are assumed to undergo biologically important size-modulations from nanorafts to microrafts. Due to the complexity of cellular membranes, model systems become important tools, especially for the investigation of the factors affecting “raft-like” L_o domain size and the search for L_o nanodomains as precursors in L_o microdomain formation. Because lipid compositional change is the primary mechanism by which a cell can alter membrane phase behavior, we studied the effect of the ganglioside GM1 concentration on the L_o/L_d lateral phase separation in PC/SM/Chol/GM1 bilayers. GM1 above 1 mol % abolishes the formation of the micrometer-scale L_o domains observed in GUVs. However, the apparently homogeneous phase observed in optical microscopy corresponds in fact, within a certain temperature range, to a L_o/L_d lateral phase separation taking place below the optical resolution. This nanoscale phase separation is revealed by fluorescence spectroscopy, including C₁₂NBD-PC self-quenching and Laurdan GP measurements, and is supported by Gaussian spectral decomposition analysis. The temperature of formation of nanoscale L_o phase domains over an L_d phase is determined, and is shifted to higher values when the GM1 content increases. A “morphological” phase diagram could be made, and it displays three regions corresponding respectively to L_o/L_d micrometric phase separation, L_o/L_d nanometric phase separation, and a homogeneous L_d phase. We therefore show that a lipid only-based mechanism is able to control the existence and the sizes of phase-separated membrane domains. GM1 could act on the line tension, “arresting” domain growth and thereby stabilizing L_o nanodomains.     

Activation of phospholipase A2 by ternary model membranes

Formation of liquid-ordered domains in model membranes can be linked to raft formation in cellular membranes. The lipid stoichiometry has a governing influence on domain formation and consequently, biochemical hydrolysis of specific lipids has the potential to remodel domain features. Activation of phospholipase A₂ (PLA₂) by ternary model membranes with three components (DOPC/DPPC/Cholesterol) can potentially change the domain structure by preferential hydrolysis of the phospholipids. Using fluorescence microscopy, this work investigates the changes in domain features that occur upon PLA₂ activation by such ternary membranes. Double-supported membranes are used, which have minimal interactions with the solid support. For membranes prepared in the coexistence region, PLA₂ induces a decrease of the liquid-disordered (L_d) phase and an increase of the liquid-ordered (L_o) phase. A striking observation is that activation by a uniform membrane in the L_d phase leads to nucleation and growth of L_o-like domains. This phenomenon relies on the initial presence of cholesterol and no PLA₂ activation is observed by membranes purely in the L_o phase. The observations can be rationalized by mapping partially hydrolyzed islands onto trajectories in the phase diagram. It is proposed that DPPC is protected from hydrolysis through interactions with cholesterol, and possibly the formation of condensed complexes. This leads to specific trajectories which can account for the observed trends. The results demonstrate that PLA₂ activation by ternary membrane islands may change the global lipid composition and remodel domain features while preserving the overall membrane integrity.     

Characterization of Horizontal Lipid Bilayers as a Model System to Study Lipid Phase Separation

Artificial lipid membranes are widely used as a model system to study single ion channel activity using electrophysiological techniques. In this study, we characterize the properties of the artificial bilayer system with respect to its dynamics of lipid phase separation using single-molecule fluorescence fluctuation and electrophysiological techniques. We determined the rotational motions of fluorescently labeled lipids on the nanosecond timescale using confocal time-resolved anisotropy to probe the microscopic viscosity of the membrane. Simultaneously, long-range mobility was investigated by the lateral diffusion of the lipids using fluorescence correlation spectroscopy. Depending on the solvent used for membrane preparation, lateral diffusion coefficients in the range D_lat = 10-25 μm²/s and rotational diffusion coefficients ranging from D_rot = 2.8 − 1.4 × 10⁷ s⁻¹ were measured in pure liquid-disordered (L_d) membranes. In ternary mixtures containing saturated and unsaturated phospholipids and cholesterol, liquid-ordered (L_o) domains segregated from the L_d phase at 23°C. The lateral mobility of lipids in L_o domains was around eightfold lower compared to those in the L_d phase, whereas the rotational mobility decreased by a factor of 1.5. Burst-integrated steady-state anisotropy histograms, as well as anisotropy imaging, were used to visualize the rotational mobility of lipid probes in phase-separated bilayers. These experiments and fluorescence correlation spectroscopy measurements at different focal diameters indicated a heterogeneous microenvironment in the L_o phase. Finally, we demonstrate the potential of the optoelectro setup to study the influence of lipid domains on the electrophysiological properties of ion channels. We found that the electrophysiological activity of gramicidin A (gA), a well-characterized ion-channel-forming peptide, was related to lipid-domain partitioning. During liquid-liquid phase separation, gA was largely excluded from L_o domains. Simultaneously, the number of electrically active gA dimers increased due to the increased surface density of gA in the L_d phase.     

Temperature induced lipid membrane restructuring and changes in nanomechanics

The naturally occurring milk sphingomyelin is of particular interest owing to its complex composition and involvement in the formation of the milk fat globule membrane (MFGM). Knowledge of membrane organization and nanomechanical stability has proved to be crucial in understanding their properties and functions. In this work, two model membrane systems composed of 1, 2 dioleoyl-sn-glycero-3-phosphocholine (DOPC), egg sphingomyelin (egg-SM) and cholesterol, and DOPC, milk sphingomyelin (milk-SM) and cholesterol were exposed to both RT and 10 °C. The morphological and nanomechanical changes were investigated using atomic force microscopy (AFM) imaging and force mapping below RT using a designed liquid cell with temperature-control. In both systems, the size and shape of SM/Chol-enriched liquid ordered domains (L_o) and DOPC-enriched liquid disordered phase (L_d) were monitored at controlled temperatures. AFM based force-mapping showed that rupture forces were consistently higher for L_o domains than L_d phases and were decreased for L_d with decreasing temperature while an increase in breakthrough force was observed in L_o domains. More interestingly, dynamic changes and defect formations in the hydrated lipid bilayers were mostly detected at low temperature, suggesting a rearrangement of lipid molecules to relieve additional tension introduced upon cooling. Noteworthy, in these model membrane systems, tension-driven defects generally heal on reheating the sample. The results of this work bring new insights to low temperature induced membrane structural reorganization and mechanical stability changes which will bring us one step closer to understand more complex systems such as the MFGM.     

Fluid domain patterns in free-standing membranes captured on a solid support

We devise a methodology to fixate and image dynamic fluid domain patterns of giant unilamellar vesicles (GUVs) at sub-optical length scales. Individual GUVs are rapidly transferred to a solid support forming planar bilayer patches. These are taken to represent a fixated state of the free standing membrane, where lateral domain structures are kinetically trapped. High-resolution images of domain patterns in the liquid-ordered (l_o) and liquid-disordered (l_d) co-existence region in the phase-diagram of ternary lipid mixtures are revealed by atomic force microscopy (AFM) scans of the patches. Macroscopic phase separation as known from fluorescence images is found, but with superimposed fluctuations in the form of nanoscale domains of the l_o and l_d phases. The size of the fluctuating domains increases as the composition approaches the critical point, but with the enhanced spatial resolution, such fluctuations are detected even deep in the coexistence region. Agreement between the area-fraction of domains in GUVs and the patches respectively, supports the assumption that the thermodynamic state of the membrane remains stable. The approach is not limited to specific lipid compositions, but could potentially help uncover lateral structures in highly complex membranes.     

Mechanism of local anesthetic-induced disruption of raft-like ordered membrane domains

BackgroundBecause ordered membrane domains, called lipid rafts, regulate activation of ion channels related to the nerve pulse, lipids rafts are thought to be a possible target for anesthetic molecules. To understand the mechanism of anesthetic action, we examined influence of representative local anesthetics (LAs); dibucaine, tetracaine, and lidocaine, on raft-like liquid-ordered (L_o)/non-raft-like liquid-disordered (L_d) phase separation.MethodsImpact of LAs on the phase separation was observed by fluorescent microscopy. LA-induced perturbation of the L_o and L_d membranes was examined by DPH anisotropy measurements. Incorporation of LAs to the membranes was examined by fluorescent anisotropy of LAs. The biding location of the LAs was indicated by small angle x-ray diffraction (SAXD).ResultsFluorescent experiments showed that dibucaine eliminated the phase separation the most effectively, followed by tetracaine and lidocaine. The disruption of the phase separation can be explained by their disordering effects on the L_o membrane. SAXD and other experiments further suggested that dibucaine's most potent perturbation of the L_o membrane is attributable to its deeper immersion and bulky molecular structure. Tetracaine, albeit immersed in the L_o membrane as deeply as dibucaine, less perturbs the L_o membrane probably because of its smaller bulkiness. Lidocaine hardly reaches the hydrophobic region, resulting in the weakest L_o membrane perturbation.ConclusionDibcaine perturbs the L_o membrane the most effectively, followed by tetracaine and lidocaine. This ranking correlates with their anesthetic potency.General significanceThis study suggests a possible mechanistic link between anesthetic action and perturbation of lipid rafts.     

Domain redistribution within ergosterol-containing model membranes in the presence of the antimicrobial compound fengycin

The cyclic lipopeptide fengycin, produced by Bacillus subtilis, exhibits its antimicrobial capabilities by altering the integrity of the cell membrane of plant pathogens. Previous work has correlated fengycin activity with membrane characteristics, such as sterol content. This work focused on the influence of fengycin on supported lipid bilayers containing varying levels of ergosterol. Total internal reflection fluorescence (TIRF) microscopy was used to visualize and distinguish ordered (L_β/L_o) and disordered (L_α/L_d) domains in the model membranes following exposure to low (50 μg) and high (500 μg) fengycin doses. Application of an initial low dose of fengycin to 0% and 3% ergosterol-containing bilayers resulted in redistribution of L_α/L_β and L_o/L_d domains, respectively, which the bilayers compensated and corrected for over time. These membranes were unable to tolerate a second 50 μg dose or a single high fengycin dose. The 6% ergosterol bilayers were able to tolerate sequential low doses of fengycin. Exposure of these bilayers to the high fengycin dose caused a decrease in the number of L_o domains, albeit less than that seen in the 0% and 3% ergosterol bilayers. Bilayers containing 12% ergosterol, exhibited the least amount of change after fengycin exposure. These were the only bilayer to exhibit an increase in area taken up by ordered domains. These results suggest fengycin may preferentially act on the L_β or L_o phase, the area in which ergosterol resides. Bilayers containing low levels of ergosterol appear to be more sensitive to the lipopeptide, suggesting ergosterol plays a role in buffering perturbations caused by fengycin.     

In Situ Determination of Structure and Fluctuations of Coexisting Fluid Membrane Domains

Biophysical understanding of membrane domains requires accurate knowledge of their structural details and elasticity. We report on a global small angle x-ray scattering data analysis technique for coexisting liquid-ordered (L_o) and liquid-disordered (L_d) domains in fully hydrated multilamellar vesicles. This enabled their detailed analysis for differences in membrane thickness, area per lipid, hydrocarbon chain length, and bending fluctuation as demonstrated for two ternary mixtures (DOPC/DSPC/CHOL and DOPC/DPPC/CHOL) at different cholesterol concentrations. L_o domains were found to be ∼10 Å thicker, and laterally up to 20 Å²/lipid more condensed than L_d domains. Their bending fluctuations were also reduced by ∼65%. Increase of cholesterol concentration caused significant changes in structural properties of L_d, while its influence on L_o properties was marginal. We further observed that temperature-induced melting of L_o domains is associated with a diffusion of cholesterol to L_d domains and controlled by L_o/L_d thickness differences.     

A detailed analysis of partial molecular volumes in DPPC/cholesterol binary bilayers

We examined the volumetric behavior of the dipalmitoylphosphatidylcholine (DPPC)/cholesterol binary bilayer system with high accuracy and more cholesterol concentrations to reveal the detailed molecular states in the liquid-disordered (L_d) phase, the liquid-ordered (L_o) phase and the gel phase. We measured the average specific volume of the binary bilayer at several temperatures by the neutral flotation method and calculated the average volume per molecule to estimate the partial molecular volumes of DPPC and cholesterol in each phase. As a result, we found that the region with intermediate cholesterol concentrations showed a more complicated behavior than expected from simple coexistence of L_d and L_o domains. We also measured fluorescence decay of trans-parinaric acid (tPA) added into the binary bilayer with more cholesterol concentrations to get further insight into the cholesterol-induced formation of the L_o phase. On the basis of these results we discuss the molecular interaction between DPPC and cholesterol molecule in the L_o phase and the manner of L_d/L_o phase coexistence.     

Effect of cytochrome c on the phase behavior of charged multicomponent lipid membranes

We studied the effect of submicromolar concentrations of cytochrome c (cyt c) on the phase behavior of ternary lipid membranes composed of charged dioleoylphosphatidylglycerol, egg sphingomyelin and cholesterol. The protein was found to induce micron-sized domains in membranes belonging to the single-fluid-phase region of the protein-free ternary mixture and, as a result, to expand the region of coexistence of liquid ordered (L_o) and liquid disordered (L_d) phases. Direct observations on individual vesicles revealed that protein adsorption increases the area of L_d domains. Measurements using a fluorescent analog of cyt c showed that the protein preferentially adsorbs onto domains belonging to the L_d phase. The adsorption was quantitatively characterized in terms of partitioning ratios between the L_d and the L_o phases. The protein was also found to induce vesicle leakage even at relatively low concentrations. In eukaryotic cells under normal physiological conditions, cyt c is localized within the intermembrane space of mitochondria. During cell apoptotis, cyt c is released into the cytosol and its adsorption to intracellular membranes may strongly perturb the lipid distribution within these membranes as suggested by our results.     

Deuterium NMR of Raft Model Membranes Reveals Domain-Specific Order Profiles and Compositional Distribution

In this report, we applied site-specifically deuterated N-stearoylsphingomyelins (SSMs) to raft-exhibiting ternary mixtures containing SSM, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol (Chol) and successfully acquired deuterium quadrupole coupling profiles of SSM from liquid-ordered (L_o) and liquid-disordered (L_d) domains. To our knowledge, this is the first report that shows detailed lipid chain dynamics separately and simultaneously obtained from coexisting L_o and L_d domains. We also found that the quadrupole profile of the L_o phase in the ternary system was almost identical to that in the SSM-Chol binary mixture, suggesting that the order profile of the binary system is essentially applicable to more complicated membrane systems in terms of the acyl chain order. We also demonstrated that ²H NMR spectroscopy, in combination with organic synthesis of deuterated components, could be used to reveal the accurate mole fractions of each component distributed in the L_o and L_d domains. As compared with the reported tie-line analysis of phase diagrams, the merit of our ²H NMR analysis is that the domain-specific compositional fractions are directly attainable without experimental complexity and ambiguity. The accurate compositional distributions as well as lipid order profiles in ternary mixtures are relevant to understanding the molecular mechanism of lipid raft formation.     

Limitations of electronic energy transfer in the determination of lipid nanodomain sizes

Even though superresolution microscopy indicates that size of plasma membrane rafts is <20 nm, those structures have never been observed. Förster resonance energy transfer (FRET) is therefore still the most powerful optical method for characterization of such domains. In this letter we investigate relation between nanodomain affinity of a donor-acceptor (D/A) pair and the detectable nanodomain size/area. We show that probes with high affinity to the liquid-ordered (L_o) phase are required for detecting domain sizes of a few nanometers, and/or domains that occupy a few percent of the bilayer area. A combination of donors and acceptors that prefer different phases is the more favorable approach. For instance, a D/A pair with the distribution constant of donors K_D = 5 and acceptors K_A = 0.01 can resolve a broad spectrum of nanodomain sizes. On the other hand, currently available donors and acceptors that prefer the same phase, either the liquid-disordered (L_d) or L_o phase, are not so convenient for determining domain sizes <20 nm. Here the detection limits of FRET experiments employing several commonly used D/A pairs have been investigated.     

Docosahexaenoic acid promotes micron scale liquid-ordered domains. A comparison study of docosahexaenoic versus oleic acid containing phosphatidylcholine in raft-like mixtures

The understanding of the functional role of the lipid diversity in biological membranes is a major challenge. Lipid models have been developed to address this issue by using lipid mixtures generating liquid-ordered (L_o)/liquid-disordered (L_d) immiscibility. The present study examined mixtures comprising Egg sphingomyelin (SM), cholesterol (chol) and phosphatidylcholine (PC) either containing docosahexaenoic (PDPC) or oleic acid (POPC). The mixtures were examined in terms of their capability to induce phase separation at the micron- and nano-scales. Fluorescence microscopy, electron spin resonance (ESR), X-ray diffraction (XRD) and calorimetry methods were used to analyze the lateral organization of the mixtures. Fluorescence microscopy of giant vesicles could show that the temperature of the micron-scale L_o/L_d miscibility is higher for PDPC than for POPC ternary mixtures. At 37 °C, no micron-scale L_o/L_d phase separation could be identified in the POPC containing mixtures while it was evident for PDPC. In contrast, a phase separation was distinguished for both PC mixtures by ESR and XRD, indicative that PDPC and POPC mixtures differed in micron vs nano domain organization. Compared to POPC, the higher line tension of the L_o domains observed in PDPC mixtures is assumed to result from the higher difference in L_o/L_d order parameter rather than hydrophobic mismatch.     

Adhesion Stabilizes Robust Lipid Heterogeneity in Supercritical Membranes at Physiological Temperature

Regions of contact between cells are frequently enriched in or depleted of certain protein or lipid species. Here, we explore a possible physical basis that could contribute to this membrane heterogeneity using a model system of a giant vesicle tethered to a planar supported bilayer. Vesicles contain coexisting liquid-ordered (L_o) and liquid-disordered (L_d) phases at low temperatures and are tethered using trace quantities of adhesion molecules that preferentially partition into one liquid phase. We find that the L_d marker DiI-C₁₂ is enriched or depleted in the adhered region when adhesion molecules partition into L_d or L_o phases, respectively. Remarkably, adhesion stabilizes an extended zone enriched or depleted of DiI-C₁₂ even at temperatures >15°C above the miscibility phase transition when membranes have compositions that are in close proximity to a critical point. A stable adhesion zone is also observed in plasma membrane vesicles isolated from living RBL-2H3 cells, and probe partitioning at 37°C is diminished in vesicles isolated from cells with altered cholesterol levels. Probe partitioning is in good quantitative agreement with predictions of the two-dimensional Ising model with a weak applied field for both types of model membranes. These studies experimentally demonstrate that large and stable domain structure can be mediated by lipids in single-phase membranes with supercritical fluctuations.     

Aggregatibacter actinomycetemcomitans leukotoxin causes activation of lymphocyte function‐associated antigen 1

Repeats‐in‐toxin leukotoxin (LtxA) produced by the oral bacterium Aggregatibacter actinomycetemcomitans kills human leukocytes in a lymphocyte function‐associated antigen 1 (LFA‐1, integrin α_L/β₂)‐dependent manner, although the mechanism for this interaction has not been identified. The LtxA internalisation by LFA‐1‐expressing cells was explored with florescence resonance energy transfer (FRET) microscopy using a cell line that expresses LFA‐1 with a cyan fluorescent protein‐tagged cytosolic α_L domain and a yellow fluorescent protein‐tagged β₂ domain. Phorbol 12‐myristate 13‐acetate activation of LFA‐1 caused transient cytosolic domain separation. However, addition of LtxA resulted in an increase in FRET, indicating that LtxA brings the cytosolic domains closer together, compared with the inactive state. Unlike activation, this effect was not transient, lasting more than 30 min. Equilibrium constants of LtxA binding to the cytoplasmic domains of both α_L and β₂ were determined using surface plasmon resonance. LtxA has a strong affinity for the cytosolic domains of both the α_L and β₂ subunits (K_d = 15 and 4.2 nM, respectively) and a significantly lower affinity for the cytoplasmic domains of other integrin α_M, α_X, and β₃ subunits (K_d = 400, 180, and 230 nM, respectively), used as controls. Peptide fragments of α_L and β₂ show that LtxA binds membrane‐proximal domain of α_L and intermediate domain of β₂.     

Effects of Lipid Composition and Phase on the Membrane Interaction of the Prion Peptide 106-126 Amide

Lipid rafts are specialized liquid-ordered (L_o) phases of the cell membrane that are enriched in sphingolipids and cholesterol (Chl), and surrounded by a liquid-disordered (L_d) phase enriched in glycerophospholipids. Lipid rafts are involved in the generation of pathological forms of proteins that are associated with neurodegenerative diseases. To investigate the effects of lipid composition and phase on the generation of pathological forms of proteins, we constructed an L_d-gel phase-separated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/sphingomyelin (from bovine brain (BSM))-supported lipid bilayer (SLB) and an L_d-L_o phase-separated POPC/BSM/Chl SLB. We used in situ time-lapse atomic force microscopy to study the interactions between these SLBs and the prion peptide K¹⁰⁶TNMKHMAGAAAAGAVVGGLG¹²⁶ (PrP106-126) amide, numbered according to the human prion-peptide sequence. Our results show that: 1), with the presence of BSM in the L_d phase, the PrP106-126 amide induces fully penetrated porations in the L_d phase of POPC/BSM SLB and POPC/BSM/Chl SLB; 2), with the presence of both BSM and Chl in the L_d phase, the PrP106-126 amide induces the disintegration of the L_d phase of POPC/BSM/Chl SLB; and 3), with the presence of both BSM and Chl in the L_o phase, PrP106-126 amide induces membrane thinning in the L_o phase of POPC/BSM/Chl SLB. These results provide comprehensive insight into the process by which the PrP106-126 amide interacts with lipid membranes.