Effect of Sucrose on Phase Behavior of Membranes in Intact Pollen of Typha latifolia L., as Measured with Fourier Transform Infrared Spectroscopy

In previous studies, we have shown that the temperature dependent vibrational frequency of the CH₂ stretch in hydrocarbons in intact pollen grains can be recorded with Fourier transform infrared spectroscopy and used to measure phase transition temperatures (T_m) in these hydrocarbons. Circumstantial evidence was provided that the major contribution to the signal seen in these samples was from membrane phospholipids, and that sucrose in the dry pollen grains reduced T_m of those phospholipids. In the current study, we clarify why a major constituent of the pollen grains, neutral lipids contained in discrete lipid droplets, does not contribute significantly to the signal. Further, we have isolated membranes from the pollen and show that T_m in the isolated membranes rises from −6°C in the hydrated membranes to 58°C when the membranes are dried without the addition of sucrose. However, when the isolated membranes are dried in the presence of increasing amounts of sucrose, T_m fell steadily, reaching a minimal value of 31°C, a figure in good agreement with that seen in the intact pollen grains. The amount of sucrose required to depress T_m maximally in these membranes is also apparently in agreement with that found in the intact pollen, suggesting that sucrose depresses T_m in the pollen.     

Phase transitions and permeability changes in dry membranes during rehydration

Dry phospholipid bilayers are known to undergo transient changes in permeability during rehydration. In this review, we present evidence from which we suggest that this permeability change is due to a gel to liquid-crystaline phase transition accompanying rehydration. If the transition is avoided, as in lipids that remain in gel phase whether dry or rehydrated, the problem of leakage during rehydration is obviated, at least in part. Further, the evidence that the transition temperature for dry bilayers can be depressed by certain sugars is discussed. Finally, we show that these principles can be extended to intact cells. Using pollen grains as a model, we have measured the transition temperature for membrane phospholipids and show that the transition is correlated with physiological measurements including permeability changes and subsequent germination. From theT _m values taken from pollen grains at different water contents, we have constructed a phase diagram for the intact pollen that has high predictive value for physiological properties.     

Physical properties of membrane fractions isolated from human platelets: implications for chilling induced platelet activation.

In previous studies, it has been suggested that chilling induced activation of human platelets is related to a lipid phase transition seen in membrane lipids. Those studies showed a single, surprisingly cooperative transition in human platelets, as determined by Fourier transform infrared (FTIR) spectroscopy, findings that are confirmed here with calorimetric measurements. Such transitions have now been studied in membrane fractions obtained from the platelets and it is reported that all fractions and purified phospholipids show similar transitions. In order to obtain these data it was necessary to develop means for separating these fractions. Therefore, a novel method for isolation and separation of dense tubular system (DTS) and plasma membranes in human platelets is described here. Lipid analysis showed that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the dominant phospholipids in both fractions, whereas cholesterol and sphingomyelin (SM) were predominantly located in the plasma membranes. Thermotropic phase transitions in the two membrane fractions, determined by differential scanning calorimetry (DSC) and FTIR spectroscopy were found to occur at about 15 degrees C, similar to the Tm of intact human platelets. These data are discussed in relation to the role of the DTS and plasma membranes in the cold-induced activation of human platelets.     

Physical properties of membrane fractions isolated from human platelets: implications for chilling induced platelet activation

In previous studies, it has been suggested that chilling induced activation of human platelets is related to a lipid phase transition seen in membrane lipids. Those studies showed a single, surprisingly cooperative transition in human platelets, as determined by Fourier transform infrared (FTIR) spectroscopy, findings that are confirmed here with calorimetric measurements. Such transitions have now been studied in membrane fractions obtained from the platelets and it is reported that all fractions and purified phospholipids show similar transitions. In order to obtain these data it was necessary to develop means for separating these fractions. Therefore, a novel method for isolation and separation of dense tubular system (DTS) and plasma membranes in human platelets is described here. Lipid analysis showed that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the dominant phospholipids in both fractions, whereas cholesterol and sphingomyelin (SM) were predominantly located in the plasma membranes. Thermotropic phase transitions in the two membrane fractions, determined by differential scanning calorimetry (DSC) and FTIR spectroscopy were found to occur at about 15 degrees C, similar to the Tm of intact human platelets. These data are discussed in relation to the role of the DTS and plasma membranes in the cold-induced activation of human platelets.     

Phase behavior of phosphatidylglycerol in spinach thylakoid membranes as revealed by 31P-NMR

Non-bilayer lipids account for about half of the total lipid content in chloroplast thylakoid membranes. This lends high propensity of the thylakoid lipid mixture to participate in different phases which might be functionally required. It is for instance known that the chloroplast enzyme violaxanthin de-epoxidase (VDE) requires a non-bilayer phase for proper functioning in vitro but direct evidence for the presence of non-bilayer lipid structures in thylakoid membranes under physiological conditions is still missing. In this work, we used phosphatidylglycerol (PG) as an intrinsic bulk lipid label for 31P-NMR studies to monitor lipid phases of thylakoid membranes. We show that in intact thylakoid membranes the characteristic lamellar signal is observed only below 20 degrees C. But at the same time an isotropic phase is present, which becomes even dominant between 14 and 28 degrees C despite the presence of fully functional large membrane sheets that are capable of generating and maintaining a transmembrane electric field. Tris-washed membranes show a similar behavior but the lamellar phase is present up to higher temperatures. Thus, our data show that the location of the phospholipids is not restricted to the bilayer phase and that the lamellar phase co-exists with a non-bilayer isotropic phase.     

Phase behavior of phosphatidylglycerol in spinach thylakoid membranes as revealed by P-NMR

Non-bilayer lipids account for about half of the total lipid content in chloroplast thylakoid membranes. This lends high propensity of the thylakoid lipid mixture to participate in different phases which might be functionally required. It is for instance known that the chloroplast enzyme violaxanthin de-epoxidase (VDE) requires a non-bilayer phase for proper functioning in vitro but direct evidence for the presence of non-bilayer lipid structures in thylakoid membranes under physiological conditions is still missing.In this work, we used phosphatidylglycerol (PG) as an intrinsic bulk lipid label for ³¹P-NMR studies to monitor lipid phases of thylakoid membranes. We show that in intact thylakoid membranes the characteristic lamellar signal is observed only below 20 °C. But at the same time an isotropic phase is present, which becomes even dominant between 14 and 28 °C despite the presence of fully functional large membrane sheets that are capable of generating and maintaining a transmembrane electric field. Tris-washed membranes show a similar behavior but the lamellar phase is present up to higher temperatures. Thus, our data show that the location of the phospholipids is not restricted to the bilayer phase and that the lamellar phase co-exists with a non-bilayer isotropic phase.     

Quick-freeze differential scanning calorimetry and saturation transfer electron spin resonance: novel techniques for assessing phase transitions in biological membranes

Quick-freeze differential scanning calorimetry (QF-DSC) and saturation transfer-electron spin resonance (ST-ESR) spectroscopy were used to study lipid gel-phase transitions in mature green tomato fruit microsomal membranes. ST-ESR of 12-doxyl methyl stearate labelled membranes proved to be reproducible and provided increased sensitivity to temperature-induced structural changes, allowing the detection of several transitions in isolated membranes (6 degrees C, 21 degrees C, 28 degrees C). QF-DSC led to the assessment of lipid gel phase transitions in isolated microsomal membranes and microsomal membrane lipids by enhancing the transition. A phase transition enthalpy of 114 J/g and an onset temperature of 29.8 degrees C were obtained for whole membranes while with isolated lipids values of 370 J/g and 19.9 degrees C were found.     

Intra- and extracellular lipid composition and associated gene expression patterns during pollen development in Brassica napus

Pollen development in angiosperms is regulated by the interaction of products contributed by both the gametophytic (haploid) and sporophytic (diploid) genomes. In entomophilous species, lipids are major products of both sporophytic and gametophytic metabolism during pollen development. Mature pollen grains of Brassica napus are shown to contain three major acyl lipid pools as follows: (i) the extracellular tryphine mainly consisting of medium-chain neutral esters; (ii) the intracellular membranes, particularly endoplasmic reticulum, mainly containing phospholipids; and (iii) the intracellular storage lipids, which are mostly triacylglycerols. This paper reports on the kinetics of accumulation of these lipid classes during pollen maturation and the expression patterns of several lipid biosynthetic genes and their protein products that are differentially regulated in developing microspores/ pollen grains (gametophyte) and tapetal cells (sporophyte) of B. napus. Detailed analysis of three members of the stearoyl-ACP desaturase (sad) gene family by Northern blotting, in situ hybridization and RT-PCR showed that the same individual genes were expressed both in gametophytic and sporophytic tissues, although under different temporal regulation. In the tapetum, maximal expression of two marker genes for lipid biosynthesis (sad and ear) occurred at a bud length of 2–3 mm, and the corresponding gene products SAD and EAR were detected by Western blotting in 3–4 mm buds, coinciding with the maximal rates of tapetal lipid accumulation. These lipids are released following tapetal cell disintegration and are relocated to form the major structural component of the extracellular tryphine layer that coats the mature pollen grain. In contrast, in developing microspores/pollen grains, maximal expression of the lipid marker genes sad, ear, acp and cyb5 was at the 3–5 mm bud stages, with the SAD and EAR gene products detected in 4–7 mm buds. This pattern of expression coincided with accumulation of the intracellular storage and membrane lipid components of pollen. These results suggest that, although the same genes may be expressed in the sporophytic tapetal cells and in gametophytic tissues, they are regulated differentially leading to the production of the various contrasting lipidic structures that are assembled together to give rise to a viable, fertile pollen grain.     

Different modes of ozone-induced lipid oxidation in Candida utilis yeast cells and isolated membrane preparations

Significant differences in the development of ozonolysis of lipids in membrane preparations and intact cells of the Candida utilis yeast were revealed. First, unlike isolated membranes, in which lipid modifications can be initiated by low ozone doses (< 0.5 micromol O3/mg protein) and develop proportionally to the treatment dose, in intact yeast cells, even the most ozone-sensitive sterols and nitrogen-containing phospholipids (phosphatidylcholine and phosphatidylethanolamine) did not undergo oxidative destruction at doses up to 6.0 micromol O3/mg protein. Second, the peculiarity of the ozone-initiated lipid modification in intact cells was that different classes of lipids exhibited different sensitivity to ozone. With an increase in the ozone dose, neutral lipids (sterols) and nitrogen-containing phospholipids (phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) were modified to a greater extent. Third, the accumulation of lipid peroxidation products upon ozone treatment of cells, in contrast to the isolated membranes, was absent at low ozone doses and was recorded only after the lethal damage. It is suggested that these differences are related to both the function of antioxidative enzymes (catalase, superoxide dismutase, peroxidase, etc.) and the difference between the structural states (i.e., stability and accessibility to oxidation) of lipids in the isolated membranes and the intact cells.     

Altered Phase Behavior in Membranes of Aging Dry Pollen May Cause Imbibitional Leakage

Aging of dry pollen has been shown to coincide with increases of free fatty acids and lysophospholipids. These compounds reduce the integrity of hydrated liposomes made from isolated pollen phospholipids but do not lead to their total destruction. However, a massive, instantaneous leakage occurs upon imbibition of dry cattail pollen (Typha latifolia) that has aged to the point of complete loss of viability. To resolve the apparent discrepancy in stability between hydrated and dry membranes, the lyotropic phase behavior of two liposome systems containing lysophospholipid (12 mol%) was studied with differential scanning calorimetry and Fourier transform infrared spectroscopy. In both systems dehydration caused phase separation of the lipids. Fourier transform infrared data concerning phase behavior of isolated membranes from aging pollen and of membranes in situ did not show phase separations, probably because the assay technique was not sufficiently sensitive to detect them. However, aging of the pollen resulted in a permanent increase in the gel-to-liquid crystalline phase transition temperature (Tm) of isolated membranes and in a broadening of the transition in situ. We conclude that the increase in Tm of hydrated membranes may be more closely related to the leakage.     

Dynamics properties of membrane proteins in native cell membranes revealed by solid-state NMR spectroscopy

Cell membranes provide an environment that is essential to the functions of membrane proteins. Cell membranes are mainly composed of proteins and highly diverse phospholipids. The influence of diverse lipid compositions of native cell membranes on the dynamics of the embedded membrane proteins has not been examined. Here we employ solid-state NMR to investigate the dynamics of E. coli Aquaporin Z (AqpZ) in its native inner cell membranes, and reveal the influence of diverse lipid compositions on the dynamics of AqpZ by comparing it in native cell membranes to that in POPC/POPG bilayers. We demonstrate that the dynamic rigidity of AqpZ generally conserves in both native cell membranes and POPC/POPG bilayers, due to its tightly packed tetrameric structure. In the gel and the liquid crystal phases of lipids, our experimental results show that AqpZ is more dynamic in native cell membranes than that in POPC/POPG bilayers. In addition, we observe that phase transitions of lipids in native membranes are less sensitive to temperature variations compared with that in POPC/POPG bilayers, which results in that the dynamics of AqpZ is less affected by the phase transitions of lipids in native cell membranes than that in POPC/POPG bilayers. This study provides new insights into the dynamics of membrane proteins in native cell membranes.     

X-ray studies on the interaction of the antimicrobial peptide gramicidin S with microbial lipid extracts: evidence for cubic phase formation

We have investigated the effect of the interaction of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of model lipid bilayer membranes generated from the total membrane lipids of Acholeplasma laidlawii B and Escherichia coli. The A. laidlawii B membrane lipids consist primarily of neutral glycolipids and anionic phospholipids, while the E. coli inner membrane lipids consist exclusively of zwitterionic and anionic phospholipids. We show that the addition of GS at a lipid-to-peptide molar ratio of 25 strongly promotes the formation of bicontinuous inverted cubic phases in both of these lipid model membranes, predominantly of space group Pn3m. In addition, the presence of GS causes a thinning of the liquid-crystalline bilayer and a reduction in the lattice spacing of the inverted cubic phase which can form in the GS-free membrane lipid extracts at sufficiently high temperatures. This latter finding implies that GS potentiates the formation of an inverted cubic phase by increasing the negative curvature stress in the host lipid bilayer. This effect may be an important aspect of the permeabilization and eventual disruption of the lipid bilayer phase of biological membranes, which appears to be the mechanism by which GS kills bacterial cells and lysis erythrocytes.     

Enveloped viruses as model membrane systems: microviscosity of vesicular stomatitis virus and host cell membranes.

The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene was used to study and compare the dynamic properties of the hydrophobic region of vesicular stomatitis virus grown on L-929 cells, plasma membrane of L-929 cells prepared by two different methods, liposomes prepared from virus lipids and plasma membrane lipids, and intact L-929 cells. The rate of penetration of the probe into the hydrophobic region of the lipid bilayer was found to be much faster in the lipid vesicle bilayer as compared with the intact membrane, but in all cases the fluorescence anisotropy was constant with time. The L-cell plasma membranes, the vesicles prepared from the lipids derived from the plasma membranes, and intact cells are found to have much lower microviscosity values than the virus or virus lipid vesicles throughout a wide range of temperatures. The microviscosity of plasma membrane and plasma membrane lipid vesicles was found to depend on the procedure for plasma membrane preparation as the membranes prepared by different methods had different microviscosities. The intact virus and liposomes prepared from the virus lipids were found to have very similar microviscosity values. Plasma membrane and liposomes prepared from plasma membrane lipids also had similar microviscosity values. Factors affecting microviscosity in natural membranes and artificially mixed lipid membranes are discussed.     

Phospholipid phase transitions in homogeneous nanometer scale bilayer discs

Nanoscale protein supported phospholipid bilayer discs, or Nanodiscs, were produced for the purpose of studying the phase transition behavior of the incorporated lipids. Nanodiscs and vesicles were prepared with two phospholipids, dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine, and the phase transition of each was analyzed using laurdan fluorescence and differential scanning calorimetry. Laurdan is a fluorescent probe sensitive to the increase of hydration in the lipid bilayer that accompanies the gel to liquid crystalline phase transition. The emission intensity profile can be used to derive the generalized polarization, a measure of the relative amount of each phase present. Differential scanning calorimetry was used to further quantitate the phase transition of the phospholipids. Both methods revealed broader transitions for the lipids in Nanodiscs compared to those in vesicles. Also, the transition midpoint was shifted 3-4 degrees C higher for both lipids when incorporated into Nanodiscs. These findings are explained by a loss of cooperativity in the lipids of Nanodiscs which is attributable to the small size of the Nanodiscs as well as the interaction of boundary lipids with the protein encircling the discs. The broad transition of the Nanodisc lipid bilayer better mimics the phase behavior of cellular membranes than vesicles, making Nanodiscs a 'native-like' lipid environment in which to study membrane associated proteins.     

Fatty-acid composition of lipids and physicochemical characteristics of membrane fractions and the membrane of endoplasmic reticulum of thymus and Pliss' lymphosarcoma

The paper is concerned with composition of neutral lipids and phospholipids, the regularity of lipid bilayer and structural reorganization of plasma membranes, and membranes of smooth and rough cell reticulum of thymus and Pliss lymphosarcoma are studied at linear and stationary growth phase. No qualitative differences are found in the fatty-acid composition of lipid membranes in normal and tumour cells. In plasma membranes of phospholipids and in membranes of smooth reticulum of tumour cells the unsaturated lipid component increases in the process of growth, the cholesterin/phospholipids ratio decreases, fluidity of the lipid bilayer diminishes and structural heterogeneity of these membranes rises while in membranes of rough reticulum the saturation of lipids increases, but the cholesterin/phospholipids ratio does not change. The temperatures of structural reorganization also does not change, which evidences for a less structural lability of membranes of rough reticulum as compared with other membranes.     

Pollen Lipidomics: Lipid Profiling Exposes a Notable Diversity in 22 Allergenic Pollen and Potential Biomarkers of the Allergic Immune Response

Background/Aim

Pollen grains are the male gametophytes that deliver sperm cells to female gametophytes during sexual reproduction of higher plants. Pollen is a major source of aeroallergens and environmental antigens. The pollen coat harbors a plethora of lipids that are required for pollen hydration, germination, and penetration of the stigma by pollen tubes. In addition to proteins, pollen displays a wide array of lipids that interact with the human immune system. Prior searches for pollen allergens have focused on the identification of intracellular allergenic proteins, but have largely overlooked much of the extracellular pollen matrix, a region where the majority of lipid molecules reside. Lipid antigens have attracted attention for their potent immunoregulatory effects. By being in close proximity to allergenic proteins on the pollen surface when they interact with host cells, lipids could modify the antigenic properties of proteins.

Methodology/Principal Findings

We performed a comparative pollen lipid profiling of 22 commonly allergenic plant species by the use of gas chromatography-mass spectroscopy, followed by detailed data mining and statistical analysis. Three experiments compared pollen lipid profiles. We built a database library of the pollen lipids by matching acquired pollen-lipid mass spectra and retention times with the NIST/EPA/NIH mass-spectral library. We detected, identified, and relatively quantified more than 106 lipid molecular species including fatty acids, n-alkanes, fatty alcohols, and sterols. Pollen-derived lipids stimulation up-regulate cytokines expression of dendritic and natural killer T cells co-culture.

Conclusions/Significance

Here we report on a lipidomic analysis of pollen lipids that can serve as a database for identifying potential lipid antigens and/or novel candidate molecules involved in allergy. The database provides a resource that facilitates studies on the role of lipids in the immunopathogenesis of allergy. Pollen lipids vary greatly among allergenic species and contain many molecules that have stimulatory or regulatory effects on immune responses.     

Chemistry and biology of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids: fluorescent probes of biological and model membranes

Lipids that are covalently labeled with the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group are widely used as fluorescent analogues of native lipids in model and biological membranes to study a variety of processes. The fluorescent NBD group may be attached either to the polar or the apolar regions of a wide variety of lipid molecules. Synthetic routes for preparing the lipids, and spectroscopic and ionization properties of these probes are reviewed in this report. The orientation of various NBD-labeled lipids in membranes, as indicated by the location of the NBD group, is also discussed. The NBD group is uncharged at neutral pH in membranes, but loops up to the surface if attached to acyl chains of phospholipids. These lipids find applications in a variety of membrane-related studies which include membrane fusion, lipid motion and dynamics, organization of lipids and proteins in membranes, intracellular lipid transfer, and bilayer to hexagonal phase transition in liposomes. Use of NBD-labeled lipids as analogues of natural lipids is critically evaluated.     

Structure and dynamics of the phosphatidylcholine and the phosphatidylethanolamine head group in L-M fibroblasts as studied by deuterium nuclear magnetic resonance.

Mouse fibroblast L-M cells were grown in tissue culture medium containing selectively deuterated choline or ethanolamine. Both compounds were incorporated into the corresponding phospholipids at levels greater than 50% thus leading to a selective deuteration of these phospholipid head groups. Choline and ethanolamine were labeled at either the alpha- or the beta-carbon atom and well-resolved deuterium and phosphorus n.m.r. spectra were obtained from intact cells, crude plasma membranes and lipid extracts, leading to the following conclusions. (i) A large fraction, if not all, of the phospholipids in the intact L-M cell membranes were organized in a liquid crystalline bilayer. (ii) The phosphoethanolamine and the phosphocholine head group conformation were found to be remarkably similar in pure lipid bilayers and in intact L-M cell membranes with the head group dipoles being oriented parallel to the membrane surface. (iii) The deuterium T1 spin lattice relaxation times fell in the range of 7-25 ms and were similar in intact L-M cells and in pure lipid model membranes, suggesting that the two head groups are not involved in strong interactions with membrane proteins. The rotational diffusion rate of the two head groups was reduced by at least a factor of 10 compared to molecules of the same size in aqueous solution. (iv) The phosphocholine head group was sensitive to the size and sign of membrane surface charges as verified in mixing experiments with charged lipids. In L-M cell membranes the phosphocholine appeared to sense an electrically neutral environment in spite of the fact that L-M cell membranes contain 10-20% negatively charged lipids.     

Temperature-induced structural transition in-situ in porcine lens — Changes observed in void size distribution

The function of mammalian ocular lens is to provide a sharp image to the retina. Accordingly, the lens needs to be transparent and minimize light scattering. To do so the lens fiber cells first loose intracellular organelles, organize the cytoplasm and arrange the fiber cell membranes. Because the fiber cells are metabolically inactive, the plasma membrane becomes the only cellular organelle and consequently, the phase behavior of these membranes determines the physiological state of the lens. Previous studies have shown that lipids extracted from the nuclear and cortical region of human lens show a temperature-induced phase transition close to the body temperature. Yet, the physiological function of this phase transition is not known, and even the presence of the phase transition in intact lenses is unknown. Positron annihilation lifetime spectroscopy (PALS) was used to characterize the sub-nanometer-sized local structure of intact porcine lens and these studies were complemented with differential scanning calorimeter and mass spectrometric analysis in extracted porcine lens lipids. Using PALS, we present evidence for the presence of a temperature-dependent structural transition centered at 35.5 °C in-situ in clear extracted porcine lenses. Further studies employing extracted lens lipids and purified egg-yolk sphingomyelin and cholesterol mixtures suggest that the nano-scale transition emerges from the phase behavior of lens lipids. Based on our results, PALS seems to be a viable method for gaining additional information on biological tissues, especially since it enables non-destructive studies on intact tissues.     

A Theoretical Model for the Association Probabilities of Saturated Phospholipids from Two-Component Bilayer Lipid Membranes

The non-random mixing of biomembrane components, especially saturated phospholipids, exhibits important consequences in molecular biology. Particularly, the distribution of lipids within natural and model membranes is strongly determined by the selective association processes. These processes of phospholipids take place due to the cooperative modes in multiparticle systems as well as the specific lipid-lipid interactions both in the hydrophobic core and in the region of the polar headgroups. We demonstrated that the investigation of the selective association processes of saturated phospholipids might contribute to the insight of the lipid domains appearance inside the bilayer membranes. The association probabilities of like-pairs and cross-pairs from a binary mixture of saturated phospholipids were tested for both parallel and anti-parallel alignments of the polar headgroups. The present model confirms the experimental evidence for saturated phospholipids to have a high tendency for association in parallel configuration of the electric dipole moments of the polar headgroups whether the cross-sectional area of the polar headgroup is in an usual range of 25-55 2. There are three major lipid domains in a binary mixture of saturated phospholipids: (i) lipid domains in non-mixed phase of the first mixture component, in parallel alignment of the polar headgroups; (ii) lipid domains in non-mixed phase of the second mixture component, in anti-parallel alignment of the polar headgroups; (iii) lipid domains in mixed phase. We think that the selective association processes of phospholipids are neither exclusively, nor only involved in promoting the lipid domains appearance through bilayer phospholipid membranes.