Formation Of Membrane Domains Research Articles

Characterization of cholesterol crystalline domains in model and biological membranes using X-ray diffraction.

In this review, we discuss the use of small-angle X-ray diffraction approaches in studying the formation of cholesterol crystalline domains in membranes derived from model and biological sources. Numerous studies have shown that monomeric cholesterol can self-associate and form immiscible, membrane-restricted domains as a result of increased membrane concentration or systematic oxidation of membrane phospholipids. These domains are observed, in an X-ray diffraction pattern, as reflections describing a unit cell periodicity of 34 Å, which is consistent with cholesterol molecules arranged in a tail-to-tail, bilayer conformation. In vascular smooth muscle cells isolated from animal models of atherosclerosis, plasmalemmal cholesterol domain formation is associated with cellular dysfunction, including the disruption of calcium transport mechanisms. Cholesterol domains are also observed in macrophages and precede the deposition of extracellular cholesterol crystals in the atheroma. We have also shown that cholesterol domains can be produced in model membranes following exposure to oxidative stress and other disease-like conditions such as hyperglycemia. By contrast, cholesterol domains appear to be essential to the normal physiology of certain cell groups such as those of the human ocular lens. Cholesterol domains are a prominent structural feature of the lens fiber cell plasma membrane where they are believed to facilitate lens tissue transparency and interfere with various mechanisms of cataract formation. These contrasting biologic effects highlight the complex role that cholesterol domains play in cell plasma membrane structure-function relationships in both health and disease.KeywordsCholesterol DomainsFiber Cell Plasma MembraneFiber CellsCalcium Transport MechanismsTulenkoThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Cholesterol selectively regulates IL-5 induced mitogen activated protein kinase signaling in human eosinophils.

Eosinophils function contributes to human allergic and autoimmune diseases, many of which currently lack curative treatment. Development of more effective treatments for eosinophil-related diseases requires expanded understanding of eosinophil signaling and biology. Cell signaling requires integration of extracellular signals with intracellular responses, and is organized in part by cholesterol rich membrane microdomains (CRMMs), commonly referred to as lipid rafts. Formation of these organizational membrane domains is in turn dependent upon the amount of available cholesterol, which can fluctuate widely with a variety of disease states. We tested the hypothesis that manipulating membrane cholesterol content in primary human peripheral blood eosinophils (PBEos) would selectively alter signaling pathways that depend upon membrane-anchored signaling proteins localized within CRMMs (e.g., mitogen activated protein kinase [MAPK] pathway), while not affecting pathways that signal through soluble proteins, like the Janus Kinase/Signal Transducer and Activator of Transcription [JAK/STAT] pathway. Cholesterol levels were increased or decreased utilizing cholesterol-chelating methyl-β-cyclodextrin (MβCD), which can either extract membrane cholesterol or add exogenous membrane cholesterol depending on whether MβCD is preloaded with cholesterol. Human PBEos were pretreated with MβCD (cholesterol removal) or MβCD+Cholesterol (MβCD+Chol; cholesterol delivery); subsequent IL-5-stimulated signaling and physiological endpoints were assessed. MβCD reduced membrane cholesterol in PBEos, and attenuated an IL-5-stimulated p38 and extracellular-regulated kinase 1/2 phosphorylation (p-p38, p-ERK1/2), and an IL-5-dependent increase in interleukin-1β (IL-1β) mRNA levels. In contrast, MβCD+Chol treatment elevated PBEos membrane cholesterol levels and basal p-p38, but did not alter IL-5-stimulated phosphorylation of ERK1/2, STAT5, or STAT3. Furthermore, MβCD+Chol pretreatment attenuated an IL-5-induced increase in cell survival at 48 hours, measured as total cellular metabolism. The reduction in cell survival following cholesterol addition despite unaltered STAT phosphorylation contradicts the current dogma in which JAK/STAT activation is sufficient to promote eosinophil survival, and suggests an additional, unidentified mechanism critically regulates IL-5-mediated human PBEos survival.

1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-rich domain formation in binary phospholipid vesicle membranes: two-dimensional nucleation and growth.

Decades of study have probed phase transitions in model phospholipid bilayers and vesicles, especially in the context of the equilibrium phase diagram. Critical to the response of vesicles to environmental triggers, to the ultimate domain morphology, and to the approach to equilibrium (or not), we present here a study of domain formation in vesicles, focusing on a mechanism by which the cooling rate, tension, and composition affect the first appearance (nucleation) and subsequent growth of solid membrane domains. Employing a popular mixed membrane model based on DOPC and DPPC (1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, respectively), we examined phase separation in giant two-component vesicles that were cooled from the one-phase fluid (Lα) region of the phase diagram into a region of fluid (Lα)-solid coexistence. At moderate and low membrane tensions, cooling produced solid DPPC-rich domains appearing as compact patches or irregular hexagons and likely with a Pβ' (ripple) arrangement. (The compact solid domains in this study differed distinctly from striped domains in vesicles of the same composition, in terms of molecular organization and conditions of first appearance during cooling.) The amounts of these solid domains were shown to adhere to the lever arm rule for a tie line on the phase diagram, with a solid composition near 95 mol % DPPC. The nucleation of the compact solid domains occurred in a short period, followed by rapid addition of ordered molecules to the nucleated domains, excluding tracer dye. The two-dimensional nucleation density of these compact solid domains (in the range of 10(-2)-10(-1) μm(-2)) was found to increase with the cooling rate (equivalent to the quench depth) with a greater than linear dependence. The 2-D nucleation density was also seen to decrease with membrane tension, presumably because membrane tension increases the line tension around a domain that opposes nucleation. A sigmoidal dependence of the nucleation density on the DPPC concentration was also found. With cooling rates in excess of ∼1 °C/min, solid domains persisted down to room temperature, likely passing from a preferred equilibrium to a local equilibrium with continued cooling. As a result of the persistence of the originally nucleated domains and the conservation of DPPC in the membrane, we observed an increasingly greater number of smaller domains with increased cooling rates. The domains in these vesicles were compact or hexagonal-shaped in contrast to flower-shaped dendritic domains in the same membrane system in a supported membrane configuration.

The Extracellular δ-Domain is Essential for the Formation of CD81 Tetraspanin Webs

CD81 is a ubiquitously expressed member of the tetraspanin family. It forms large molecular platforms, so-called tetraspanin webs that play physiological roles in a variety of cellular functions and are involved in viral and parasite infections. We have investigated which part of the CD81 molecule is required for the formation of domains in the cell membranes of T-cells and hepatocytes. Surprisingly, we find that large CD81 platforms assemble via the short extracellular δ-domain, independent from a strong primary partner binding and from weak interactions mediated by palmitoylation. The δ-domain is also essential for the platforms to function during viral entry. We propose that, instead of stable binary interactions, CD81 interactions via the small δ-domain, possibly involving a dimerization step, play the key role in organizing CD81 into large tetraspanin webs and controlling its function.

Biophysical Implications of Sphingosine Accumulation in Membrane Properties at Neutral and Acidic pH

Sphingosine (Sph) is a simple lipid involved in the regulation of several biological processes. When accumulated in the late endosomal/lysosomal compartments, Sph causes changes in ion signaling and membrane trafficking, leading to the development of Niemann-Pick disease type C. Little is known about Sph interaction with other lipids in biological membranes; however, understanding the effect of Sph in the physical state of membranes might provide insights into its mode of action. Using complementary established fluorescence approaches, we show that Sph accumulation leads to the formation of Sph-enriched gel domains in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and POPC/sphingomyelin (SM)/cholesterol (Chol) model membranes. These domains are more easily formed in membrane models mimicking the neutral pH plasma membrane environment (PM) as compared to the acidic lysosomal membrane environment (LM), where higher Sph concentrations (or lower temperatures) are required. Electrophoretic light scattering measurements further revealed that in PM-raft models (POPC/SM/Chol), Sph is mainly neutral, whereas in LM models, the positive charge of Sph leads to electrostatic repulsion, reducing the Sph ability to form gel domains. Thus, formation of Sph-enriched domains in cellular membranes might be strongly regulated by Sph charge.

Sphingolipid symmetry governs membrane lipid raft structure

Lipid domain formation in membranes underlies the concept of rafts but their structure is controversial because the key role of cholesterol has been challenged. The configuration of glycosphingolipid receptors for agonists, bacterial toxins and enveloped viruses in plasma membrane rafts appears to be an important factor governing ligand binding and infectivity but the details are as yet unresolved. I have used X-ray diffraction methods to examine how cholesterol affects the distribution of glycosphingolipid in aqueous dispersions of an equimolar mixture of cholesterol and egg-sphingomyelin containing different proportions of glucosylceramide from human extracts. Three coexisting liquid-ordered bilayer structures are observed at 37°C in mixtures containing up to 20mol% glycosphingolipid. All the cholesterol was sequestered in one bilayer with the minimum amount of sphingomyelin (33mol%) to prevent formation of cholesterol crystals. The other two bilayers consisted of sphingomyelin and glucosylceramide. Asymmetric molecular species of glucosylceramide with N-acyl chains longer than 20 carbons form an equimolar complex with sphingomyelin in which the glycosidic residues are arranged in hexagonal array. Symmetric molecular species mix with sphingomyelin in proportions less than equimolar to form quasicrystalline bilayers. When the glycosphingolipid exceeds equimolar proportions with sphingomyelin cholesterol is incorporated into the structure and formation of a gel phase of glucosylceramide is prevented. The demonstration of particular structural features of ceramide molecular species combined with the diversity of sugar residues of glycosphingolipid classes paves the way for a rational approach to understanding the functional specificity of lipid rafts and how they are coupled across cell membranes.

Interaction of an Antitumor Peptide with Lipids of the Cancer Plasma Membrane - Formation of Membrane Domains and Influence of Cholesterol

R-DIM-P-LF11-322, derived from the cationic human host defense peptide lactoferricin, exhibits selective antitumor activity against several human cancer cell types. We have shown that cancer cells expose the negatively charged phosphatidylserine (PS) on the outer leaflet of the plasma membrane (1), which in healthy cells only comprises neutral lipids as phosphatidylcholine (PC) or cholesterol. Therefore the membrane interaction of R-DIM-P-LF11-322 with membrane mimics composed of mixtures of PS, PC and cholesterol was studied. Lipid analysis of normal melanocytes and melanoma revealed a slight increase of PC and cholesterol in the cancer lipid extracts.Tryptophan fluorescence showed that the peptide penetrated only in the cancer mimics containing ratios of PS. Addition of cholesterol slightly reduced the affinity of the peptide.Calorimetric studies with peptide and mixed liposomes of PS and PC indicated complete perturbation of the PS fraction leaving PC unaffected. Interestingly upon addition of cholesterol the peptide induced separation into PC and PS enriched domains, but seemed to be slightly reduced in its effect.Studies using giant unilamellar vesicles showed that the peptide interacts on cancer model membranes slightly stronger in the absence of cholesterol by formation of extremely deformed and destroyed vesicles. In the presence of cholesterol the peptide however still induced shrinking of the vesicles and formation of many small PS domains.In summary the antitumor peptide strongly interacts only with its target PS on cancer cells. However, cholesterol seems to play a role in the modulation of membrane domain formation and reducing the membrane affinity, which may represent a mechanism for cancer cells to protect themselves against antitumor peptides.Acknowledgment: Austrian Science Foundation FWF (grant no: P24608)(1) Riedl et al. BBA 1808: 2638-2645, 2011.

Eisosomes and Plasma Membrane Domain Formation

A vast body of evidence coming from different microscopy techniques has been instrumental in concluding the long debate on whether biological membranes presented lateral segregation of proteins and lipids. Currently, the existence of membrane domains in both eukaryotes and prokaryotes is common ground. However, the mechanisms that sustain membrane domain formation and maintenance remain largely unknown. Our work is focus on the study of eisosomes, recently discovered plasma membrane domains in S. cerevisiae. We showed that Pil1 and Lsp1, the major proteinaceous components of eisosomes, are able to form self-assemblies that bind and curve membranes both in vivo and in vitro. We also showed that Lsp1 and Pil1 membrane-sculpting abilities are associated with the generation and organization of membrane domains (1,2). Thus, our current work supports the hypothesis that a mechanism for membrane eisosome domain formation is membrane curvature generation directed by Pil1-Lsp1 assemblies. To address this hypothesis we are applying Fluorescence Correlation Spectroscopy and Number and Brightness Analysis to describe the dynamics of Pil1 and Lsp1 in vivo. We are studying the oligomeric state and concentration of these proteins in the cytoplasm soluble fraction and in the plasma membrane. Also we plan to study the kinetic parameters of Pil1, Lsp1 and plasma membrane interactions during eisosome biogenesis and also during stationary phase.

Membrane Regulation and Signal Transduction by Annexin A5

Annexins are an abundant family of proteins well known for their Ca2+-dependent membrane binding ability. Though they've been implicated in numerous membrane processes their method of action and signal integration is largely unknown. Here we propose a mechanism for annexin to integrate information regarding Ca2+ concentration and resting distribution of lipid and cholesterol in one leaflet, and transduce that information across the bilayer. In a system poised near random distribution, such as lipids of the inner leaflet of the plasma membrane, order can be imposed through even weak interactions. We've previously shown annexin's weak Ca2+-independent membrane binding acts to create a cooperative Ca2+ binding response. In this work we demonstrate not only annexin's ability to respond to and integrate information of membrane composition; but also its ability change and regulate that distribution. Protein interactions with membranes can induce membrane order and domain formation if the protein has specificity for individual lipids distributed non-ideally, or even randomly throughout the membrane. A protein that preferentially binds a free component of the membrane alters the amount of that component within other membrane domains. Annexin perturbs membrane distribution through its preferential binding and sequestration of phosphatidylserine headgroups (PS). Cholesterol perturbs phospholipid physical properties and preferentially forms complexes with PS. Then the local sequestering of PC-cholesterol complexes, and annexin-induced membrane domain formation upon Ca2+ influx, can act as the driving force for cholesterol redistribution across the bilayer. Using ITC as well as fluorescent probes such as DHE (cholesterol analogue) and carboxyfluorescein encapsulating LUVs. We show annexin's binding of cholesterol-PS complexes induces differential Ca2+ binding sites, and redistributes cholesterol throughout the bilayer without disrupting membrane permeability. Together this suggests that annexin has the capacity to redistribute cholesterol across the bilayer in a calcium-ion and membrane composition dependent manner.

Solubilization of Giant Vesicles Composed of Erythrocyte Lipid Extracts and of Ternary Lipid Mixtures by Triton X-100

Detergents are widely used as solubilizing agents of biological membranes. Detergent resistant membranes, called DRMs, are frequently obtained and share similarities in composition and properties with the so-called lipid rafts and with the liquid-ordered phase (Lo) of model membranes. Here we use optical and fluorescence microscopy to study the solubilization process of giant unilamellar vesicles (GUVs) made of erythrocyte lipid extracts (erythro-GUVs) by the commonly used detergent Triton X-100. For comparison, ternary lipid mixtures of POPC, sphingomyelin (SM) and cholesterol are also investigated. Erythro-GUVs do not exhibit macroscopic phase separation, as seen by the homogenous distribution of a fluorescence probe that preferentially partition into the fluid (Ld) phase. However, contact with Triton X-100 immediately causes formation of micrometer-sized bright domains, typical of Lo/Ld phase separation. Then, at Triton X-100 concentrations above its cmc, the fluid part is first detached from the original vesicle and then completely solubilized by Triton X-100. The insoluble vesicle left is only faintly fluorescent and appears to be in the Lo phase. Typically, one third of the erythro-GUV surface area is solubilized. Solubilization of POPC:SM:chol GUVs in a certain composition range follow the same sequence of events. Our results show that Triton X-100 promotes formation of macroscopic domains in both biological and biomimetic membranes, and that the partial solubilization occurs after phase separation, suggesting that the composition of the insoluble membrane fraction is modulated by the choice of the detergent used. Financial support: CNPq, FAPESP and INCT-FCx.

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.

Adhesion-Induced Domain Formation in Multicomponent Membranes

Lipid vesicles containing a relatively small number of molecular components provide simple model systems for cell membranes, which contain a large number of lipid and protein components. Such multicomponent membranes can undergo phase separation into two coexisting liquid phases and, thus, exhibit liquid domains within a liquid matrix. The associated phase diagrams depend on a number of parameters such as the overall membrane composition as well as on pH and temperature of the aqueous medium. Here, we address the influence of an adhesive substrate surface onto the phase behavior of the membranes. Specifically, we study giant unilamellar vesicles (GUVs) that can be prepared from different lipid mixtures and have sizes similar to those of cells. We control the adhesion by electrostatic membrane-surface interactions using a novel setup which allows tuning of the adhesion energy by an externally applied potential; see Fig. 1. With our single-vesicle approach, we can explore the phase behaviour of multicomponent membranes for different adhesion energies. One important objective is to identify distinct composition regimes for the phase separation in the bound and unbound part of the adhering vesicle membranes.Figure -Side views (xz scans) of a one-component GUV (about 60 µm in diameter) obtained by confocal microscopy. Fluorescence of the GUV membrane is shown in green and the adhesion surface as a red line. Left: no applied potential. Right: 1V applied potential. The change in adhesion strength can be clearly seen.

Ligand binding and complex formation of galectin-3 is modulated by pH variations

Galectin-3-dependent clusters or lattices are formed at the surface as well as in distinct organelles of eukaryotic cells. Incorporation into membrane proximal networks can fix glycoproteins within subcellular domains or sort them into distinct transport pathways. In the present paper we analysed the effect of acidification on the sugar binding and self-oligomerization of galectin-3. Using a fluorescence anisotropy assay we measured decreasing galectin-3 affinities to the blood group antigen GalNAcα1-3(Fucα1-2)Galβ1-4Glc under low pH conditions. Binding to the strong interaction partner N-acetyl-D-lactosamine was also lost at pH 5.0, whereas the less efficient ligand lactose was still able to bind. This indicates that variations in the binding specificity to distinct glycans can be observed by altering the pH. The formation of galectin-3-based complexes by interaction with the multivalent glycoproteins asialofetuin or transferrin was also obliterated at acidic pH and the ligand-binding affinity itself was modulated by oligomerization of the lectin. When galectin-3 was added to giant plasma membrane vesicles from the apical surface of epithelial cells, pH modulation could generate or eliminate the formation of membrane domains enriched with p75(NTR) (neurotrophin receptor p75). In conclusion, the results of the present study suggest that the formation and composition of galectin-3 networks can be fine-tuned by changes in the environmental pH.

Transient domain formation in membrane-bound organelles undergoing maturation

The membrane components of cellular organelles have been shown to segregate into domains as the result of biochemical maturation. We propose that the dynamical competition between maturation and lateral segregation of membrane components regulates domain formation. We study a two-component fluid membrane in which enzymatic reaction irreversibly converts one component into another and phase separation triggers the formation of transient membrane domains. The maximum domain size is shown to depend on the maturation rate as a power law similar to the one observed for domain growth with time in the absence of maturation, despite this time dependence not being verified in the case of irreversible maturation. This control of domain size by enzymatic activity could play a critical role in regulating exchange between organelles or within compartmentalized organelles such as the Golgi apparatus.

A 2H Solid-State NMR Study of Lipid Clustering by Cationic Antimicrobial and Cell-Penetrating Peptides in Model Bacterial Membranes

Domain formation in bacteria-mimetic membranes due to cationic peptide binding was recently proposed based on calorimetric data. We now use 2H solid-state NMR to critically examine the presence and absence of domains in bacterial membranes containing zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) lipids. Chain-perdeuterated POPE and POPG are used in single-component membranes, binary POPE/POPG (3:1) membranes, and membranes containing one of four cationic peptides: two antimicrobial peptides (AMPs) of the β-hairpin family of protegrin-1 (PG-1), and two cell-penetrating peptides (CPPs), HIV TAT and penetratin. 2H quadrupolar couplings were measured to determine the motional amplitudes of POPE and POPG acyl chains as a function of temperature. Homogeneously mixed POPE/POPG membranes should give the same quadrupolar couplings for the two lipids, whereas the presence of membrane domains enriched in one of the two lipids should cause distinct 2H quadrupolar couplings that reflect different chain disorder. At physiological temperature (308 K), we observed no or only small coupling differences between POPE and POPG in the presence of any of the cationic peptides. However, around ambient temperature (293 K), at which gel- and liquid-crystalline phases coexist in the peptide-free POPE/POPG membrane, the peptides caused distinct quadrupolar couplings for the two lipids, indicating domain formation. The broad-spectrum antimicrobial peptide PG-1 ordered ∼40% of the POPE lipids while disordering POPG. The Gram-negative selective PG-1 mutant, IB549, caused even larger differences in the POPE and POPG disorder: ∼80% of POPE partitioned into the ordered phase, whereas all of the POPG remained in the disordered phase. In comparison, TAT rigidified POPE and POPG similarly in the binary membrane at ambient temperature, indicating that TAT does not cause dynamic heterogeneity but interacts with the membrane with a different mechanism. Penetratin maintained the POPE order but disordered POPG, suggesting moderate domain separation. These results provide insight into the extent of domain formation in bacterial membranes and the possible peptide structural requirements for this phenomenon.

1,2-Naphthoquinone Stimulates Lipid Peroxidation and Cholesterol Domain Formation in Model Membranes

Naphthalene induces cataract formation through the accumulation of its reactive metabolite, 1,2-naphthoquinone (1,2-NQ), in the ocular lens. 1,2-NQ increases lens protein oxidation and disrupts fiber cell membrane function; however, the association of these effects with changes in membrane structure is not understood. The goal of this study was to determine the direct effects of 1,2-NQ on membrane lipid oxidation and structural organization. Iodometric approaches were used to measure the effects of naphthalene and 1,2-NQ on lipid hydroperoxide (LOOH) formation in model membranes composed of cholesterol and dilinoleoylphosphatidylcholine. Membrane samples were prepared at various cholesterol-to-phospholipid mole ratios and subjected to autoxidation at 37°C for 48 hours in the absence or presence of either agent alone (0.1-5.0 μM) or in combination with vitamin E. Small-angle x-ray diffraction was used to measure the effects of naphthalene and 1,2-NQ on membrane structure before and after exposure to oxidative stress. 1,2-NQ increased LOOH formation by 250% (P < 0.001) and 350% (P < 0.001) at 1.0 and 5.0 μM, respectively, whereas naphthalene decreased LOOH levels by 25% (P < 0.01) and 10% (NS). The pro-oxidant effect of 1,2-NQ was inversely affected by membrane cholesterol enrichment and completely blocked by vitamin E. 1,2-NQ also increased cholesterol domain formation by 360% in membranes exposed to oxidative stress; however, no significant changes in membrane lipid organization were observed with naphthalene under the same conditions. These data suggest a novel mechanism for naphthalene-induced cataract, facilitated by the direct effects of 1,2-NQ on lipid peroxidation and cholesterol domain formation.

Microdomains in the membrane landscape shape antigen-presenting cell function

The plasma membrane of immune cells is a highly organized cell structure that is key to the initiation and regulation of innate and adaptive immune responses. It is well-established that immunoreceptors embedded in the plasma membrane have a nonrandom spatial distribution that is important for coupling to components of intracellular signaling cascades. In the last two decades, specialized membrane microdomains, including lipid rafts and TEMs, have been identified. These domains are preformed structures ("physical entities") that compartmentalize proteins, lipids, and signaling molecules into multimolecular assemblies. In APCs, different microdomains containing immunoreceptors (MHC proteins, PRRs, integrins, among others) have been reported that are imperative for efficient pathogen recognition, the formation of the immunological synapse, and subsequent T cell activation. In addition, recent work has demonstrated that tetraspanin microdomains and lipid rafts are involved in BCR signaling and B cell activation. Research into the molecular mechanisms underlying membrane domain formation is fundamental to a comprehensive understanding of membrane-proximal signaling and APC function. This review will also discuss the advances in the microscopy field for the visualization of the plasma membrane, as well as the recent progress in targeting microdomains as novel, therapeutic approach for infectious and malignant diseases.

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.'

Relationship between CYP1A2 Localization and Lipid Microdomain Formation as a Function of Lipid Composition

Cytochrome P450 (P450) function requires the interaction of P450 and NADPH-cytochrome P450 reductase (CPR) in membranes, and is frequently studied using reconstituted systems composed solely of phosphatidylcholine. There is increasing evidence that other endoplasmic reticulum (ER) lipids can affect P450 structure, activity, and interactions with CPR. Some of these lipid effects have been attributed to the formation of organized liquid-ordered (l(o)) domains. The goal of this study was to determine if l(o) domains were formed in P450 reconstituted systems mimicking the ER membrane. CYP1A2, when incorporated in "ER-like" lipid vesicles, displayed detergent insolubility after treatment with Brij 98 and centrifugation in a sucrose gradient. Lipid probes were employed to identify domain formation in both ER-like vesicles and model membranes known to form l(o) domains. Changes in fluorescence resonance energy transfer (FRET) using an established donor/acceptor FRET pair in both ER-like and model l(o)-forming systems demonstrated the coexistence of l(o)- and liquid-disordered domains as a function of cholesterol and sphingomyelin content. Similarly, 6-dodecanoyl-2-dimethylaminonaphthalene (laurdan), a probe that reports on membrane organization, showed that cholesterol and sphingomyelin increased membrane order. Finally, brominated-phosphatidylcholine allowed for monitoring of the location of both CPR and CYP1A2 within the l(o) regions of ER-like systems. Taken together, the results demonstrate that ER-like vesicles generate microdomains, and both CYP1A2 and CPR predominantly localize into l(o) membrane regions. Probe fluorescent responses suggest that lipid microdomains form in these vesicles whether or not enzymes are included in the reconstituted systems. Thus, it does not appear that the proteins are critical for stabilizing l(o) domains.

Lipid Segregation and Membrane Budding Induced by the Peripheral Membrane Binding Protein Annexin A2*

The formation of dynamic membrane microdomains is an important phenomenon in many signal transduction and membrane trafficking events. It is driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Here we analyzed the ability of one peripherally associated membrane protein, annexin A2 (AnxA2), to induce the formation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-rich domains in giant unilamellar vesicles (GUVs) of complex lipid composition. AnxA2 is a cytosolic protein that can bind PI(4,5)P2 and other acidic phospholipids in a Ca(2+)-dependent manner and that has been implicated in cellular membrane dynamics in endocytosis and exocytosis. We show that AnxA2 binding to GUVs induces lipid phase separation and the recruitment of PI(4,5)P2, cholesterol and glycosphingolipids into larger clusters. This property is observed for the full-length monomeric protein, a mutant derivative comprising the C-terminal protein core domain and for AnxA2 residing in a heterotetrameric complex with its intracellular binding partner S100A10. All AnxA2 derivatives inducing PI(4,5)P2 clustering are also capable of forming interconnections between PI(4,5)P2-rich microdomains of adjacent GUVs. Furthermore, they can induce membrane indentations rich in PI(4,5)P2 and inward budding of these membrane domains into the lumen of GUVs. This inward vesiculation is specific for AnxA2 and not shared with other PI(4,5)P2-binding proteins such as the pleckstrin homology (PH) domain of phospholipase Cδ1. Together our results indicate that annexins such as AnxA2 can efficiently induce membrane deformations after lipid segregation, a mechanism possibly underlying annexin functions in membrane trafficking.