Membrane Lateral Organization Research Articles

A biomimetic platform to study the interactions of bioelectroactive molecules with lipid nanodomains.

In this work, we developed a biomimetic platform where the study of membrane associated redox processes and high-resolution imaging of lipid nanodomains can be both performed, based on a new functional gold modification, l-cysteine self-assembled monolayer. This monolayer proved to be ideal for the preparation of defect-free planar supported lipid bilayers (SLBs) where nanodomains with height difference of ∼1.5 nm are clearly resolved by atomic force microscopy. Single and multicomponent lipid compositions were used, leading to the formation of different phases and domains mimicking the lateral organization of cellular membranes, and in all cases stable and continuous bilayers were obtained. These platforms were tested toward the interaction with bioelectroactive molecules, the antioxidant quercetin, and the hormone epinephrine. Despite the weak interaction detected between epinephrine and lipid bilayers, our biomimetic interface was able to sense the redox process of membrane-bound epinephrine, obtain its surface concentration (9.36 × 10(-11) mol/cm(2) for a fluid bilayer), and estimate a mole fraction membrane/water partition coefficient (Kp) from cyclic voltammetric measurements (1.13 × 10(4) for a fluid phase membrane). This Kp could be used to quantitatively describe the minute changes observed in the photophysical properties of epinephrine intrinsic fluorescence upon its interaction with liposome suspensions. Moreover, we showed that the lipid membrane stabilizes epinephrine structure, preventing its oxidation, which occurs in neutral aqueous solution, and that epinephrine partition and mobility in membranes depends on lipid phase, expanding our knowledge on hormone membrane interactions.

Hydrophobic compounds reshape membrane domains.

Cell membranes have a complex lateral organization featuring domains with distinct composition, also known as rafts, which play an essential role in cellular processes such as signal transduction and protein trafficking. In vivo, perturbations of membrane domains (e.g., by drugs or lipophilic compounds) have major effects on the activity of raft-associated proteins and on signaling pathways, but they are difficult to characterize because of the small size of the domains, typically below optical resolution. Model membranes, instead, can show macroscopic phase separation between liquid-ordered and liquid-disordered domains, and they are often used to investigate the driving forces of membrane lateral organization. Studies in model membranes have shown that some lipophilic compounds perturb membrane domains, but it is not clear which chemical and physical properties determine domain perturbation. The mechanisms of domain stabilization and destabilization are also unknown. Here we describe the effect of six simple hydrophobic compounds on the lateral organization of phase-separated model membranes consisting of saturated and unsaturated phospholipids and cholesterol. Using molecular simulations, we identify two groups of molecules with distinct behavior: aliphatic compounds promote lipid mixing by distributing at the interface between liquid-ordered and liquid-disordered domains; aromatic compounds, instead, stabilize phase separation by partitioning into liquid-disordered domains and excluding cholesterol from the disordered domains. We predict that relatively small concentrations of hydrophobic species can have a broad impact on domain stability in model systems, which suggests possible mechanisms of action for hydrophobic compounds in vivo.

The Mechanism of Collapse of Heterogeneous Lipid Monolayers

Collapse of homogeneous lipid monolayers is known to proceed via wrinkling/buckling, followed by folding into bilayers in water. For heterogeneous monolayers with phase coexistence, the mechanism of collapse remains unclear. Here, we investigated collapse of lipid monolayers with coexisting liquid-liquid and liquid-solid domains using molecular dynamics simulations. The MARTINI coarse-grained model was employed to simulate monolayers of ∼80 nm in lateral dimension for 10–25 μs. The monolayer minimum surface tension decreased in the presence of solid domains, especially if they percolated. Liquid-ordered domains facilitated monolayer collapse due to the spontaneous curvature induced at a high cholesterol concentration. Upon collapse, bilayer folds formed in the liquid (disordered) phase; curved domains shifted the nucleation sites toward the phase boundary. The liquid (disordered) phase was preferentially transferred into bilayers, in agreement with the squeeze-out hypothesis. As a result, the composition and phase distribution were altered in the monolayer in equilibrium with bilayers compared to a flat monolayer at the same surface tension. The composition and phase behavior of the bilayers depended on the degree of monolayer compression. The monolayer-bilayer connection region was enriched in unsaturated lipids. Percolation of solid domains slowed down monolayer collapse by several orders of magnitude. These results are important for understanding the mechanism of two-to-three-dimensional transformations in heterogeneous thin films and the role of lateral organization in biological membranes. The study is directly relevant for the function of lung surfactant, and can explain the role of nanodomains in its surface activity and inhibition by an increased cholesterol concentration.

The lateral membrane organization and dynamics of myelin proteins PLP and MBP are dictated by distinct galactolipids and the extracellular matrix.

In the central nervous system, lipid-protein interactions are pivotal for myelin maintenance, as these interactions regulate protein transport to the myelin membrane as well as the molecular organization within the sheath. To improve our understanding of the fundamental properties of myelin, we focused here on the lateral membrane organization and dynamics of peripheral membrane protein 18.5-kDa myelin basic protein (MBP) and transmembrane protein proteolipid protein (PLP) as a function of the typical myelin lipids galactosylceramide (GalC), and sulfatide, and exogenous factors such as the extracellular matrix proteins laminin-2 and fibronectin, employing an oligodendrocyte cell line, selectively expressing the desired galactolipids. The dynamics of MBP were monitored by z-scan point fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS), while PLP dynamics in living cells were investigated by circular scanning FCS. The data revealed that on an inert substrate the diffusion rate of 18.5-kDa MBP increased in GalC-expressing cells, while the diffusion coefficient of PLP was decreased in sulfatide-containing cells. Similarly, when cells were grown on myelination-promoting laminin-2, the lateral diffusion coefficient of PLP was decreased in sulfatide-containing cells. In contrast, PLP's diffusion rate increased substantially when these cells were grown on myelination-inhibiting fibronectin. Additional biochemical analyses revealed that the observed differences in lateral diffusion coefficients of both proteins can be explained by differences in their biophysical, i.e., galactolipid environment, specifically with regard to their association with lipid rafts. Given the persistence of pathological fibronectin aggregates in multiple sclerosis lesions, this fundamental insight into the nature and dynamics of lipid-protein interactions will be instrumental in developing myelin regenerative strategies.

The Effect of Hydrophobic Matching between Lipids and Transmembrane Peptides on Sterol Bilayer Affinity

Lipid self-organization is believed to be essential for shaping the lateral structure of membranes, but it is becoming increasingly clear that also membrane proteins can be involved in the maintenance of membrane architecture. Cholesterol is thought to be important for the lateral organization of eukaryotic cell membranes and has also been implicated to take part in the sorting of cellular transmembrane proteins. It is therefore of interest to study the influence of lipid-protein interactions on membrane trafficking to find out how transmembrane proteins influence the lateral sorting of cholesterol in phospholipid bilayers. We have measured the equilibrium partitioning of the fluorescent cholesterol analog cholestatrienol between large unilamellar vesicles and methyl-β-cyclodextrin to determine the effect of hydrophobic matching on the affinity of sterols for phospholipid bilayers. The sterol partitioning was measured in 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers with and without the peptides WALP19, WALP23 or WALP27. The results showed that hydrophobic matching will affect the affinity of the sterol for the bilayers. Stronger sterol binding to the bilayers was achieved by an increase in positive hydrophobic mismatch (except in extreme situations), and a large negative hydrophobic mismatch decreased the affinity of the sterol for the bilayer. Peptide insertion into the phospholipid bilayers was also observed to depend on hydrophobic matching. In conclusion, the results showed that hydrophobic matching can affect lipid-protein interactions in a way that may facilitate the formation of lateral domains in cell membranes. This may well be of importance in membrane trafficking.

The Effect of Membrane-To-Domain Thickness Mismatch in Phase Separation Ternary Lipid Systems as a Function of Vesicle Size

Cellular membranes are no longer viewed as a homogeneous mix of lipids and proteins, but rather as having distinct lipid domains, so-called “rafts”, which are key for many biological processes. Much work devoted to understanding the actual mechanisms that drives lateral organization in cell membranes has been done on model membrane systems. This approach has given great insight into the formation of lipid rafts because in simple ternary mixtures with a saturated lipid, an unsaturated lipid and cholesterol, a region of liquid-liquid coexistence was found, with one of the liquid phases rich in cholesterol and saturated lipids, the finger-print of rafts. However, there is still no clear understanding of the molecular parameters that drives phase separation. Recently a new curvature hypothesis proposed a correlation between composition, leaflet coupling and emulsion-like induced curvature to predict phase separation and the formation of domains. To explore these ideas, we recently studied the phase behavior of well-researched phase separating ternary mixtures: dDPPC-DOPC-Cholesterol (1:1:1) and dDMPC-DOPC-Cholesterol (1:1:1), in 30nm vesicles using Small Angle Neutron Scattering. dDMPC is a 14-carbon long lipid while dDPPC is 16-carbon long lipid. As temperature was lowered, the system with dDPPC showed excess scattering, whereas the system with dDMPC showed no scattering even below its melting temperature. These results are interesting when compared to the previously established work where both systems show phase separation in GUVs as seen by fluorescent microscopy. Phase separation behavior differences as a result of variations to the tail length of the saturated lipids in our vesicles with high curvature gives insight into the role of local curvature (emulsion-like effects) at the domain-membrane interface.

Asymmetry Determines the Effect of Ceramides on Model Membranes. In Natural Membranes Too?

Ceramides can dramatically influence the lateral organization of biological membranes. In particular, ceramide-induced alterations of protein-lipid domains can be involved in several cellular processes, ranging from senescence to immune response. In this context, an important role is played by the length of the fatty acid bound to the sphingosine moiety. Asymmetric, heterogeneous ceramides, with more than 20 or less than 16 carbon atoms in the fatty acyl chain, in fact exert diverging effects in vivo if compared to their symmetric counterparts. In this work, we investigated the role of ceramide asymmetry and heterogeneity in model membranes showing raft-like phase separation, using a combination of fluorescence imaging, atomic force microscopy, fluorescence correlation spectroscopy and differential scanning calorimetry. We show that ceramide produced enzymatically from natural mixtures of sphingomyelin can dramatically alter the mixing behaviour of proteins and lipids in the membrane, inducing a homogenization of the bilayer. Furthermore, we characterized the physical properties of coexisting lipid phases at equilibrium in membranes with varying ceramide content, emphasizing the differences between symmetric-homogeneous and asymmetric-heterogeneous ceramides. While symmetric ceramides always produce enhanced order, asymmetric ceramides display a more complex behavior similar to that of cholesterol. Our results might help contribute to a more precise understanding of the rearrangements induced by different kinds of ceramide generation in cellular membranes.

Nanomechanical spectroscopy of synthetic and biological membranes.

We report that atomic force microscopy based high-speed nanomechanical analysis can identify components of complex heterogeneous synthetic and biological membranes from the measured spectrum of nanomechanical properties. We have investigated phase separated ternary lipid bilayers and purple membranes of Halobacterium salinarum. The nanomechanical spectra recorded on these samples identify all membrane components, some of which are difficult to resolve in conventional phase images. This non-destructive approach can aid the design of synthetic lipid bilayers and studies lateral organization of complex heterogeneous cellular membranes.

Divalent Cation- and Cholesterol-Induced Perturbation in Lipid Lateral Organizations and Polyphosphoinositide-Protein Interactions

Phosphatidylinositol 4,5-bisphosphate (PIP2) controls many important cellular events. A major challenge in understanding how PIP2 function in vivo is to define its physical state and lateral organization in cell membranes. The hypothesis that PIP2 forms nano-sized clusters in the presence of intracellular divalent cations by electrostatic interactions was examined in model membranes with or without cholesterol-mediated phase segregation. Additional studies show how such membrane reorganization alters the effects of PIP2 on the target proteins gelsolin and DrrA.Comparison between experimental and numerical phase diagrams suggests that a simplified electrostatic model can predict Ca2+-driven formation of PIP2 clusters, but cannot account for the difference between Ca2+ and Mg2+ in condensing PIP2-containing membranes. Differences among Ca2+, Mg2+ and multivalent polyamines in membrane condensing were revealed experimentally and related to differences in their dehydration enthalpies. Ca2+-induced perturbation of PPI-protein interactions was assayed by monolayer insertion studies using a PI4P-binding protein, DrrA. Taking advantage of its unique biphasic effect on monolayer surface pressure, in which specific insertion can be isolated from non-specific adsorption, we show that Ca2+ suppresses the specific insertion of DrrA in a concentration-dependent manner. The perturbation of PIP2-protein interactions induced by cholesterol-mediated phase segregation was probed by measuring the inhibition of gelsolin's actin filament severing activity by PIP2-containing vesicles. Cholesterol-mediated phase segregation enhances the inhibition of gelsolin by PIP2, and this effect correlates with changes in membrane ordering. This result suggests that PIP2-protein interaction depends not only on global PIP2 concentrations but also on PIP2 lateral distribution without changes in lipid synthesis or degradation. The results of this work shed light on the links between PIP2 signaling and dynamic local response at the cell membrane/cytoskeletal interface.

Polystyrene Nanoparticles Perturb Lipid Membranes.

Polystyrene is abundant in marine debris. Like most synthetic polymers, it degrades very slowly, producing smaller and smaller particles easily ingested by wildlife. The presence of plastic microscopic particles in fish and marine wildlife is massive and well documented, but its impact on cellular activity is not understood. Biological activity generally requires interaction with biological membranes, but this is difficult to study at the molecular scale in vivo. Here we use coarse-grained molecular simulations to determine the effect of nanosized polystyrene (PS) particles on the properties of model biological membranes. We find that PS nanoparticles permeate easily into lipid membranes. Dissolved in the membrane core, PS chains alter membrane structure, significantly reduce molecular diffusion, and soften the membrane. Moreover, PS severely affects membrane lateral organization by stabilizing raft-like domains. Changes in membrane properties and lateral organization can severely affect the activity of membrane proteins and thereby cellular function.

Visualizing the Molecular Timing of a Physiological Decision at the Nanoscale

Mast cells are critical players in numerous diseases, including allergy, asthma, infectious disease, cancer, and even many central nervous system disorders such as autism, anxiety, and multiple sclerosis (1,2). In conjunction with these numerous roles, mast cells are found in almost every tissue in the human body. Many of the molecular elements crucial to mast cell function, such as cytoskeletal involvement and calcium signaling, are also essential to signaling in a variety of cell types, including neurons and T cells. The article by Shelby et al. (3) in this issue of the Biophysical Journal demonstrates what we believe to be the first superresolution imaging of living mast cells, where an important cellular decision (related to a physiological response) is made at the molecular level through the lateral organization of the high-affinity IgE receptor FceRI. This molecular decision determines whether the cell will degranulate, releasing histamine, serotonin, and other effectors that lead to a host of downstream consequences. Upon antigen-induced aggregation of IgE-bound FceRI receptors, mast cell signaling results in a tyrosine phosphorylation cascade, leading to the activation of phospholipase C. In turn, inositol 1,4,5-triphosphate is produced and binds to its receptor in the endoplasmic reticulum, which activates Ca2+ influx into the cytosol, which then leads to degranulation (4). Shelby et al. (3) show ground-breaking superresolution imaging of IgE receptors in living cells using stochastic optical reconstruction microscopy (5,6), and find a dramatic reorganization of receptor molecules visualized with nanometer resolution as a function of time after stimulation. Their work is an excellent application of superresolution to membrane lateral organization: it includes careful quantification of membrane receptor clustering and diffusion, and it highlights how both spatial and dynamic information can be obtained with single-molecule localization-based superresolution methods in live cells. Furthermore, Shelby et al. (3) demonstrate an advance in our understanding of an important biological system. Importantly, this work demonstrates not just the properties of membrane clustering, but the relationships among molecular dynamics, nanoscale clustering (at length scales not previously accessible), and (crucially) cellular function. The authors are able to separate the effects of stimulation on both receptor mobility and clustering. Somewhat surprisingly, reduction in receptor mobility and assembly of receptors into clusters do not occur simultaneously. Rather, on short timescales (within 2 min of stimulation), receptor mobility reduces without strong clustering, and this occurs before the functional response of Ca2+ mobilization. Then, at ∼5 min after stimulation, the receptors reorganize into ∼70 nm clusters with ∼100 receptors each. It would be extremely difficult to carry out these studies without the use of localization microscopy: electron microscopy provides outstanding resolution, but is generally incompatible with living cells, and conventional fluorescence imaging would not reach the necessary resolution (i.e., <70 nm) or be able to count molecules. Thus, this work is a substantial biological advance that highlights the advantages of localization-based superresolution microscopy for addressing certain kinds of biological questions. Furthermore, the ability of these methods to obtain high-resolution images and to quantify molecular dynamics is a powerful combination (7–9). For example, the authors were able to determine that single receptors showed slower, more confined diffusion in regions of high receptor density—a connection between molecular mobility and spatial distribution even for receptors that are not yet strongly clustered. This finding is in contrast to the properties of hemagglutinin from influenza, another membrane-associated protein, which shows lateral confinement and clustering but only weak dependence of mobility on HA density (10). The ability to observe such differences between cellular and viral membrane proteins is only possible because of the ability to obtain both spatial and dynamic information at the molecular scale. These detailed observations are crucial to our development of a more general understanding of cell membrane organization. Several models of membrane organization now incorporate actin and/or tubulin in their explanations of membrane clustering and dynamics (11,12). Interestingly, the authors’ evidence for lateral confinement of receptors is reminiscent of the lateral confinement of a number of membrane-associated molecules, and prior evidence of actin’s role in membrane biology during mast cell signaling (13) has been obtained. These findings together strongly motivate further investigation of the nanoscale role of the cytoskeleton (14) in the observed phenomena. These illuminating findings set the stage for future superresolution investigations into the molecular details of signaling in this important biological system. It will be interesting to observe interactions among these receptors, their associated kinases, actin, and other important players in the plasma membrane with superresolution microscopy, and Shelby et al. (3) demonstrate that it can be done successfully. The powerful capabilities of live-cell super-resolution microscopy provide a promising path to a unified molecular model of membrane proteins, lipids, signaling, and the cytoskeleton.

Lateral organization of biological membranes

The lateral organization of biological membranes is of great importance in many biological processes, both for the formation of specific structures such as super-complexes and for function as observed in signal transduction systems. Over the last years, AFM studies, particularly of bacterial photosynthetic membranes, have revealed that certain proteins are able to segregate into functional domains with a specific organization. Furthermore, the extended non-random nature of the organization has been suggested to be important for the energy and redox transport properties of these specialized membranes. In the work reported here, using a coarse-grained Monte Carlo approach, we have investigated the nature of interaction potentials able to drive the formation and segregation of specialized membrane domains from the rest of the membrane and furthermore how the internal organization of the segregated domains can be modulated by the interaction potentials. These simulations show that long-range interactions are necessary to allow formation of membrane domains of realistic structure. We suggest that such possibly non-specific interactions may be of great importance in the lateral organization of biological membranes in general and in photosynthetic systems in particular. Finally, we consider the possible molecular origins of such interactions and suggest a fundamental role for lipid-mediated interactions in driving the formation of specialized photosynthetic membrane domains. We call these lipid-mediated interactions a 'lipophobic effect.'

The role of MPP1/p55 and its palmitoylation in resting state raft organization in HEL cells

Here we show the crucial role of MPP1 in lateral membrane ordering/organization in HEL cells (derived from erythroid precursors). Biochemical analyses showed that inhibition of MPP1 palmitoylation or silencing of the MPP1 gene led to a dramatic decrease in the DRM fraction. This was accompanied by a reduction of membrane order as shown by fluorescence-lifetime imaging microscopy (FLIM) analyses. Furthermore, MPP1 knockdown significantly affects the activation of MAP-kinase signaling via raft-dependent RTK (receptor tyrosine kinase) receptors, indicating the importance of MPP1 for lateral membrane organization. In conclusion, palmitoylation of MPP1 appears to be at least one of the mechanisms controlling lateral organization of the erythroid cell membrane. Thus, this study, together with our recent results on erythrocytes, reported elsewhere (Łach et al., J. Biol. Chem., 2012, 287, 18974–18984), points to a new role for MPP1 and presents a novel linkage between membrane raft organization and protein palmitoylation.

Computer simulations of the phase separation in model membranes

We used computer simulations to investigate the properties of model lipid membranes with coexisting phases. This is relevant for understanding lipid-lipid interactions underlying lateral organization in biological membranes. Molecular dynamics simulations with the MARTINI coarse-grained force field were employed to study lipid bilayers -40 nm in lateral dimension on a 20 micros time scale. The simulations retain near atomic-level detail and lipid chemical specificity, and allow formation of multiple domains of tens of nanometers in size. Using ternary lipid mixtures of saturated and unsaturated lipids and cholesterol, we reproduced the coexistence of the Lalpha/gel phases and the Lo/Ld phases. Phase transformation proceeded by either nucleation or spinodal decomposition. The properties of coexisting phases were characterized in detail, including partial lipid areas, composition, phase boundary and domain registry, based on Voronoi tessellation. We investigated variations of these properties with temperature and surface tension, and compared them to our recent simulations of lipid monolayers of the same size and composition. We found substantial overlap in bilayer and monolayer properties. Increasing the temperature in bilayers produced similar effects as increasing the surface tension in monolayers. This information can be used for interpreting experimental data on model membranes.

Molecular View of Phase Coexistence in Model Membranes

We used computer simulations to investigate phase transformations in lipid monolayers. This is important for understanding lipid-lipid interactions underlying lateral organization in biological membranes, and the role of phase coexistence in the regulation of surface tension by lung surfactant. Molecular dynamics simulations with the coarse-grained force field MARTINI were employed to achieve large length (∼80 nm in lateral dimension) and time (tens of microseconds) scales. Lipid mixtures containing saturated and unsaturated lipids and cholesterol were investigated under varying surface tension and temperature. We reproduced compositional lipid de-mixing and transformation into liquid-expanded (LE) and liquid-condensed (LC) phases, and into liquid-ordered (Lo) and liquid-disordered (Ld) phases. Transformation proceeded via either nucleation and growth, or spinodal decomposition, with distinct coarsening kinetics. Nucleation rate and growth exponents were calculated. Partial lipid areas and phase composition showed a different dependence on surface tension. The domain boundary length increased and the line tension decreased with reducing surface tension. Domains of Lo phase manifested spontaneous curvature at low surface tensions. The surface viscosity of monolayers with phase coexistence increased due to domain re-organization under shear. In the Lo/Ld mixture, strong compositional fluctuations were observed at higher temperatures. Monolayer collapse occurred in the disordered phase (LE or Ld), which then transferred into bilayer folds and monolayer-bilayer connection. Domains of coexisting phased either increased or reduced monolayer stability. We also investigated lipid bilayers of the same composition. Decreasing surface tension in monolayers and temperature in bilayers had similar effects on the properties of coexisting phases.

Sterol affinity for phospholipid bilayers is influenced by hydrophobic matching between lipids and transmembrane peptides

Lipid self-organization is believed to be essential for shaping the lateral structure of membranes, but it is becoming increasingly clear that also membrane proteins can be involved in the maintenance of membrane architecture. Cholesterol is thought to be important for the lateral organization of eukaryotic cell membranes and has also been implicated to take part in the sorting of cellular transmembrane proteins. Hence, a good starting point for studying the influence of lipid–protein interactions on membrane trafficking is to find out how transmembrane proteins influence the lateral sorting of cholesterol in phospholipid bilayers. By measuring equilibrium partitioning of the fluorescent cholesterol analog cholestatrienol between large unilamellar vesicles and methyl-β-cyclodextrin the effect of hydrophobic matching on the affinity of sterols for phospholipid bilayers was determined. Sterol partitioning was measured in 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers with and without WALP19, WALP23 or WALP27 peptides. The results showed that the affinity of the sterol for the bilayers was affected by hydrophobic matching. An increasing positive hydrophobic mismatch led to stronger sterol binding to the bilayers (except in extreme situations), and a large negative hydrophobic mismatch decreased the affinity of the sterol for the bilayer. In addition, peptide insertion into the phospholipid bilayers was observed to depend on hydrophobic matching. In conclusion, the results showed that hydrophobic matching can affect lipid–protein interactions in a way that may facilitate the formation of lateral domains in cell membranes. This could be of importance in membrane trafficking.

Modulation of MHC Binding by Lateral Association of TCR and Coreceptor

The structure of a T cell receptor (TCR) and its affinity for cognate antigen are fixed, but T cells regulate binding sensitivity through changes in lateral membrane organization. TCR microclusters formed upon antigen engagement participate in downstream signaling. Microclusters are also found 3–4 days after activation, leading to enhanced antigen binding upon rechallenge. However, others have found an almost complete loss of antigen binding four days after T cell activation, when TCR clusters are present. To resolve these contradictory results, we compared binding of soluble MHC-Ig dimers by transgenic T cells stimulated with a high (100 μM) or low (100 fM) dose of cognate antigen. Cells activated by a high dose of peptide bound sixfold lower amounts of CD8-dependent ligand Kb-SIY than cells activated by a low dose of MHC/peptide. In contrast, both cell populations bound a CD8-independent ligand Ld-QL9 equally well. Consistent with the differences between binding of CD8-dependent and CD8-independent peptide/MHC, Förster resonance energy transfer (FRET) measurements of molecular proximity reported little nanoscale association of TCR with CD8 (16 FRET units) compared to their association on cells stimulated by low antigen dose (62 FRET units). Loss of binding induced by changes in lateral organization of TCR and CD8 may serve as a regulatory mechanism to avoid excessive inflammation and immunopathology in response to aggressive infection.

GPU-Based Massive Parallel Kawasaki Kinetics in the Dynamic Monte Carlo Simulations of Lipid Nanodomains.

Multicomponent lipid membranes in the liquid phase exhibit dynamic lateral heterogeneities which play an important role in specific cell membrane functions. A GPU-based parallel algorithm for two-dimensional lattice Dynamic Monte Carlo simulations of nanodomain formation in binary lipid membranes was developed and tested. Speedups of up to 50-times over CPU-based calculations were achieved, and simulations employing lattices of up to 1800 × 1800 sites were performed. The existence of large nonregular lipid domains of sizes up to 160 nm was demonstrated. This reveals the necessity to employ lattices of at least ∼900 × 900 sites to study the lateral lipid organization in complex lipid membranes.

Palmitoylation of MPP1 (Membrane-palmitoylated Protein 1)/p55 Is Crucial for Lateral Membrane Organization in Erythroid Cells

S-Acylation of proteins is a ubiquitous post-translational modification and a common signal for membrane association. The major palmitoylated protein in erythrocytes is MPP1, a member of the MAGUK family and an important component of the ternary complex that attaches the spectrin-based skeleton to the plasma membrane. Here we show that DHHC17 is the only acyltransferase present in red blood cells (RBC). Moreover, we give evidence that protein palmitoylation is essential for membrane organization and is crucial for proper RBC morphology, and that the effect is specific for MPP1. Our observations are based on the clinical cases of two related patients whose RBC had no palmitoylation activity, caused by a lack of DHHC17 in the membrane, which resulted in a strong decrease of the amount of detergent-resistant membrane (DRM) material. We confirmed that this loss of detergent-resistant membrane was due to the lack of palmitoylation by treatment of healthy RBC with 2-bromopalmitic acid (2-BrP, common palmitoylation inhibitor). Concomitantly, fluorescence lifetime imaging microscopy (FLIM) analyses of an order-sensing dye revealed a reduction of membrane order after chemical inhibition of palmitoylation in erythrocytes. These data point to a pathophysiological relationship between the loss of MPP1-directed palmitoylation activity and perturbed lateral membrane organization.

Cholesterol Superlattice Modulates CA4P Release from Liposomes and CA4P Cytotoxicity on Mammary Cancer Cells

Liposomal drugs are a useful alternative to conventional drugs and hold great promise for targeted delivery in the treatment of many diseases. Most of the liposomal drugs on the market or under clinical trials include cholesterol as a membrane stabilizing agent. Here, we used liposomal CA4P, an antivascular drug, to demonstrate that cholesterol content can actually modulate the release and cytotoxicity of liposomal drugs in a delicate and predictable manner. We found that both the rate of the CA4P release from the interior aqueous compartment of the liposomes to the bulk aqueous phase and the extent of the drug's cytotoxicity undergo a biphasic variation, as large as 50%, with liposomal cholesterol content at the theoretically predicted Cr, e.g., 22.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol % cholesterol for maximal superlattice formation. It appears that at Cr, CA4P can be released from the liposomes more readily than at non-Cr, probably due to the increased domain boundaries between superlattice and nonsuperlattice regions, which consequently results in increased cytotoxicity. The idea that the increased domain boundaries at Cr would facilitate the escape of molecules from membranes was further supported by the data of dehydroergosterol transfer from liposomes to MβCD. These results together show that the functional importance of sterol superlattice formation in liposomes can be propagated to distal targeted cells and reveal a new, to our knowledge, mechanism for how sterol content and membrane lateral organization can control the release of entrapped or embedded molecules in membranes.