Phospholipid exchange shows insulin receptor activity is supported by both the propensity to form wide bilayers and ordered raft domains

Insulin receptor (IR) is a membrane tyrosine kinase that mediates the response of cells to insulin. IR activity has been shown to be modulated by changes in plasma membrane lipid composition, but the properties and structural determinants of lipids mediating IR activity are poorly understood. Here, using efficient methyl-alpha-cyclodextrin mediated lipid exchange, we studied the effect of altering plasma membrane outer leaflet phospholipid composition upon the activity of IR in mammalian cells. After substitution of endogenous lipids with lipids having an ability to form liquid ordered (Lo) domains (sphingomyelins) or liquid disordered (Ld) domains (unsaturated phosphatidylcholines (PCs)), we found that the propensity of lipids to form ordered domains is required for high IR activity. Additional substitution experiments using a series of saturated PCs showed that IR activity increased substantially with increasing acyl chain length, which increases both bilayer width and the propensity to form ordered domains. Incorporating purified IR into alkyl maltoside micelles with increasing hydrocarbon lengths also increased IR activity, but more modestly than by increasing lipid acyl chain length in cells. These results suggest that the ability to form Lo domains as well as wide bilayer width contributes to increased IR activity. Inhibition of phosphatases showed that some of the lipid dependence of IR activity upon lipid structure reflected protection from phosphatases by lipids that support Lo domain formation. These results are consistent with a model in which a combination of bilayer width and ordered domain formation modulates IR activity via IR conformation and accessibility to phosphatases.     

Effect of the structure of lipids favoring disordered domain formation on the stability of cholesterol-containing ordered domains (lipid rafts): identification of multiple raft-stabilization mechanisms

Despite the importance of lipid rafts, commonly defined as liquid-ordered domains rich in cholesterol and in lipids with high gel-to-fluid melting temperatures (T_m), the rules for raft formation in membranes are not completely understood. Here, a fluorescence-quenching strategy was used to define how lipids with low T_m, which tend to form disordered fluid domains at physiological temperatures, can stabilize ordered domain formation by cholesterol and high-T_m lipids (either sphingomyelin or dipalmitoylphosphatidylcholine). In bilayers containing mixtures of low-T_m phosphatidylcholines, cholesterol, and high-T_m lipid, the thermal stability of ordered domains decreased with the acyl-chain structure of low-T_m lipids in the following order: diarachadonyl > diphytanoyl > 1-palmitoyl 2-docosahexenoyl = 1,2 dioleoyl = dimyristoleoyl = 1-palmitoyl, 2-oleoyl (PO). This shows that low-T_m lipids with two acyl chains having very poor tight-packing propensities can stabilize ordered domain formation by high-T_m lipids and cholesterol. The effect of headgroup structure was also studied. We found that even in the absence of high-T_m lipids, mixtures of cholesterol with PO phosphatidylethanolamine (POPE) and PO phosphatidylserine (POPS) or with brain PE and brain PS showed a (borderline) tendency to form ordered domains. Because these lipids are abundant in the inner (cytofacial) leaflet of mammalian membranes, this raises the possibility that PE and PS could participate in inner-leaflet raft formation or stabilization. In bilayers containing ternary mixtures of PO lipids, cholesterol, and high-T_m lipids, the thermal stability of ordered domains decreased with the polar headgroup structure of PO lipids in the order PE > PS > phosphatidylcholine (PC). Analogous experiments using diphytanoyl acyl chain lipids in place of PO acyl chain lipids showed that the stabilization of ordered lipid domains by acyl chain and headgroup structure was not additive. This implies that it is likely that there are two largely mutually exclusive mechanisms by which low-T_m lipids can stabilize ordered domain formation by high-T_m lipids and cholesterol: 1), by having structures resulting in immiscibility of low-T_m and high-T_m lipids, and 2), by having structures allowing them to pack tightly within ordered domains to a significant degree.     

Partitioning of pyrene-labeled phospho- and sphingolipids between ordered and disordered bilayer domains

Here we have studied how the length of the pyrene-labeled acyl chain (n) of a phosphatidylcholine, sphingomyelin, or galactosylceramide affects the partitioning of these lipids between 1), gel and fluid domains coexisting in bovine brain sphingomyelin (BB-SM) or BB-SM/spin-labeled phosphatidylcholine (PC) bilayers or 2), between liquid-disordered and liquid-ordered domains in BB-SM/spin-labeled PC/cholesterol bilayers. The partitioning behavior was deduced either from modeling of pyrene excimer/monomer ratio versus temperature plots, or from quenching of the pyrene monomer fluorescence by spin-labeled PC. New methods were developed to model excimer formation and pyrene lipid quenching in segregated bilayers. The main result is that partition to either gel or liquid-ordered domains increased significantly with increasing length of the labeled acyl chain, probably because the pyrene moiety attached to a long chain perturbs these ordered domains less. Differences in partitioning were also observed between phosphatidylcholine, sphingomyelin, and galactosylceramide, thus indicating that the lipid backbone and headgroup-specific properties are not severely masked by the pyrene moiety. We conclude that pyrene-labeled lipids could be valuable tools when monitoring domain formation in model and biological membranes as well as when assessing the role of membrane domains in lipid trafficking and sorting.     

Progressive ordering with decreasing temperature of the phospholipids of influenza virus

Using linewidth and spinning sideband intensities of lipid hydrocarbon chain resonances in proton magic angle spinning NMR spectra, we detected the temperature-dependent phase state of naturally occurring lipids of intact influenza virus without exogenous probes. Increasingly, below 41 degrees C ordered and disordered lipid domains coexisted for the viral envelope and extracts thereof. At 22 degrees C much lipid was in a gel phase, the fraction of which reversibly increased with cholesterol depletion. Diffusion measurements and fluorescence microscopy independently confirmed the existence of gel-phase domains. Thus the existence of ordered regions of lipids in biological membranes is now demonstrated. Above the physiological temperatures of influenza infection, the physical properties of viral envelope lipids, regardless of protein content, were indistinguishable from those of the disordered fraction. Viral fusion appears to be uncorrelated to ordered lipid content. Lipid ordering may contribute to viral stability at lower temperatures, which has recently been found to be critical for airborne transmission.     

Domain coupling in asymmetric lipid bilayers

Biological membranes are heterogeneous assemblies of lipids, proteins, and cholesterol that are organized as asymmetric bimolecular leaflets of lipids with embedded proteins. Modulated by the concentration of cholesterol lipids and proteins may segregate into two or more liquid phases with different physical properties that can coexist in the same membrane. In this review, we summarize recent advances on how this situation can be recreated in a supported bilayer format and how this system has been used to demonstrate the induction of ordered lipid domains in lipid compositions that are typical for the inner leaflet by lipid compositions that are typical for the outer leaflet of mammalian plasma membranes. Proteins are shown to differentially target such induced inner leaflet domains.     

Domain coupling in asymmetric lipid bilayers

Biological membranes are heterogeneous assemblies of lipids, proteins, and cholesterol that are organized as asymmetric bimolecular leaflets of lipids with embedded proteins. Modulated by the concentration of cholesterol lipids and proteins may segregate into two or more liquid phases with different physical properties that can coexist in the same membrane. In this review, we summarize recent advances on how this situation can be recreated in a supported bilayer format and how this system has been used to demonstrate the induction of ordered lipid domains in lipid compositions that are typical for the inner leaflet by lipid compositions that are typical for the outer leaflet of mammalian plasma membranes. Proteins are shown to differentially target such induced inner leaflet domains.     

Displacement of sterols from sterol/sphingomyelin domains in fluid bilayer membranes by competing molecules

The formation of sterol and palmitoyl sphingomyelin enriched ordered domains in a fluid bilayer was examined using domain selective fluorescent reporter molecules (cholestatrienol and trans-parinaric acid containing lipids) together with a quencher molecule in the fluid phase. The aim of the study was to explore how stable the ordered domains were and how different, biologically interesting, membrane intercalators could affect domain stability and sterol distribution between domains. We show that sterols easily can be displaced from ordered domains by a variety of saturated, single- and double-chain membrane intercalators with a small polar group as a common denominator. Of the two-chain intercalators examined, both palmitoyl ceramide and palmitoyl dihydroceramide were effective in displacing sterols from ordered domains. Of the single-chain intercalators, hexadecanol and hexadecyl amide displaced the sterol from sterol/sphingomyelin domains, whereas palmitic acid, sphingosine and sphinganine failed to do so. All molecules examined stabilized the sphingomyelin-rich domains, as reported by trans-parinaric-sphingomyelin and by scanning calorimetry. Parallels between the displacement of sterol from ordered domains in our model membrane system and the ability of the above mentioned molecules to alter the chemical activity and distribution of sterols in biological membranes are discussed.     

Displacement of sterols from sterol/sphingomyelin domains in fluid bilayer membranes by competing molecules

The formation of sterol and palmitoyl sphingomyelin enriched ordered domains in a fluid bilayer was examined using domain selective fluorescent reporter molecules (cholestatrienol and trans-parinaric acid containing lipids) together with a quencher molecule in the fluid phase. The aim of the study was to explore how stable the ordered domains were and how different, biologically interesting, membrane intercalators could affect domain stability and sterol distribution between domains. We show that sterols easily can be displaced from ordered domains by a variety of saturated, single- and double-chain membrane intercalators with a small polar group as a common denominator. Of the two-chain intercalators examined, both palmitoyl ceramide and palmitoyl dihydroceramide were effective in displacing sterols from ordered domains. Of the single-chain intercalators, hexadecanol and hexadecyl amide displaced the sterol from sterol/sphingomyelin domains, whereas palmitic acid, sphingosine and sphinganine failed to do so. All molecules examined stabilized the sphingomyelin-rich domains, as reported by trans-parinaric-sphingomyelin and by scanning calorimetry. Parallels between the displacement of sterol from ordered domains in our model membrane system and the ability of the above mentioned molecules to alter the chemical activity and distribution of sterols in biological membranes are discussed.     

Glycosylation induces shifts in the lateral distribution of cholesterol from ordered towards less ordered domains

Several studies have indicated the involvement of steryl glycosides in the cellular stress response. In this work, we have compared the effect of 1-O-cholesteryl-beta-d-glucoside, 1-O-cholesteryl-beta-d-galactoside and cholesterol on the properties of glycerophospholipid and sphingolipid bilayers. The studies were performed in order to gain insight into the change in membrane properties that would follow upon the glycosylation of cholesterol in cells subjected to stress. DPH anisotropy measurements indicated that the cholesteryl glycosides (10-40 mol%) increased the order of the hydrophobic region of a POPC bilayer almost as efficiently as cholesterol. In a PSM bilayer, the cholesteryl glycosides were however shown to be much less effective compared to cholesterol in ordering the hydrocarbon chain region at temperatures above the gel to liquid-crystalline phase transition. Fluorescence quenching analysis of multicomponent lipid bilayers demonstrated that the cholesteryl glycosides, in contrast to cholesterol, were unable to stabilize ordered domains rich in PSM against temperature-induced dissociation. When the sterols were incorporated into bilayers composed of both POPC and PSM, the cholesteryl glycosides showed a higher propensity, compared to cholesterol, to influence the endothermal component representing the melting of POPC-rich domains, as determined by differential scanning calorimetry. Taken together, the results indicate that the glycosylation of cholesterol diminishes the ability of the sterol to reside in lateral domains constituted by membrane lipids having highly ordered hydrocarbon chains.     

Thermotropic behavior and lateral distribution of very long chain sphingolipids

Sphingolipids containing very long acyl chains are abundant in certain specialized tissues and minor components of plasma membranes in most mammalian cells. There are cellular processes in which these sphingolipids are required, and the function seems to be mediated through sphingolipid-rich membrane domains. This study was conducted to explore how very long acyl chains of sphingolipids influence their lateral distribution in membranes. Differential scanning calorimetry showed that 24:0- and 24:1-sphingomyelins, galactosylceramides and glucosylceramides exhibited complex thermotropic behavior and partial miscibility with palmitoyl sphingomyelin. The T_m was decreased by about 20 °C for all 24:1-sphingolipids compared to the corresponding 24:0-sphingolipids. The ability to pack tightly with ordered and extended acyl chains is a necessity for membrane lipids to partition into ordered domains in membranes and thus the 24:1-sphingolipids appeared less likely to do so. Fluorescence quenching measurements showed that the 24:0-sphingolipids formed ordered domains in multicomponent membranes, both as the only sphingolipid and mixed with palmitoyl sphingomyelin. These domains had a high packing density which appeared to hinder the partitioning of sterols into them, as reported by the fluorescent cholesterol analog cholestatrienol. 24:0-SM was, however, better able to accommodate sterol than the glycosphingolipids. The 24:1-sphingolipids could, depending on head group structure, either stabilize or disrupt ordered sphingolipid/cholesterol domains. We conclude that very long chain sphingolipids, when present in biological membranes, may affect the physical properties of or the distribution of sterols between lateral domains. It was also evident that not only the very long acyl chain but also the specific molecular structure of the sphingolipids was of importance for their membrane properties.     

The influence of membrane lipid structure on plasma membrane Ca2+ -ATPase activity

Lipid composition and Ca(2+)-ATPase activity both change with age and disease in many tissues. We explored relationships between lipid composition/structure and plasma membrane Ca(2+)-ATPase (PMCA) activity. PMCA was purified from human erythrocytes and was reconstituted into liposomes prepared from human ocular lens membrane lipids and synthetic lipids. Lens lipids were used in this study as a model for naturally ordered lipids, but the influence of lens lipids on PMCA function is especially relevant to the lens since calcium homeostasis is vital to lens clarity. Compared to fiber cell lipids, epithelial lipids exhibited an ordered to disordered phase transition temperature that was 12 degrees C lower. Reconstitution of PMCA into lipids was essential for maximal activity. PMCA activity was two to three times higher when the surrounding phosphatidylcholine molecules contained acyl chains that were ordered (stiff) compared to disordered (fluid) acyl chains. In a completely ordered lipid hydrocarbon chain environment, PMCA associates more strongly with the acidic lipid phosphatidylserine in comparison to phosphatidylcholine. PMCA associates much more strongly with phosphatidylcholine containing disordered hydrocarbon chains than ordered hydrocarbon chains. PMCA activity is influenced by membrane lipid composition and structure. The naturally high degree of lipid order in plasma membranes such as those found in the human lens may serve to support PMCA activity. The absence of PMCA activity in the cortical region of human lenses is apparently not due to a different lipid environment. Changes in lipid composition such as those observed with age or disease could potentially influence PMCA function.     

Lipid rafts reconstituted in model membranes

One key tenet of the raft hypothesis is that the formation of glycosphingolipid- and cholesterol-rich lipid domains can be driven solely by characteristic lipid-lipid interactions, suggesting that rafts ought to form in model membranes composed of appropriate lipids. In fact, domains with raft-like properties were found to coexist with fluid lipid regions in both planar supported lipid layers and in giant unilamellar vesicles (GUVs) formed from 1) equimolar mixtures of phospholipid-cholesterol-sphingomyelin or 2) natural lipids extracted from brush border membranes that are rich in sphingomyelin and cholesterol. Employing headgroup-labeled fluorescent phospholipid analogs in planar supported lipid layers, domains typically several microns in diameter were observed by fluorescence microscopy at room temperature (24 degrees C) whereas non-raft mixtures (PC-cholesterol) appeared homogeneous. Both raft and non-raft domains were fluid-like, although diffusion was slower in raft domains, and the probe could exchange between the two phases. Consistent with the raft hypothesis, GM1, a glycosphingolipid (GSL), was highly enriched in the more ordered domains and resistant to detergent extraction, which disrupted the GSL-depleted phase. To exclude the possibility that the domain structure was an artifact caused by the lipid layer support, GUVs were formed from the synthetic and natural lipid mixtures, in which the probe, LAURDAN, was incorporated. The emission spectrum of LAURDAN was examined by two-photon fluorescence microscopy, which allowed identification of regions with high or low order of lipid acyl chain alignment. In GUVs formed from the raft lipid mixture or from brush border membrane lipids an array of more ordered and less ordered domains that were in register in both monolayers could reversibly be formed and disrupted upon cooling and heating. Overall, the notion that in biomembranes selected lipids could laterally aggregate to form more ordered, detergent-resistant lipid rafts into which glycosphingolipids partition is strongly supported by this study.     

Molecular organization in striated domains induced by transmembrane alpha-helical peptides in dipalmitoyl phosphatidylcholine bilayers

Transmembrane (TM) alpha-helical peptides with neutral flanking residues such as tryptophan form highly ordered striated domains when incorporated in gel-state 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers and inspected by atomic force microscopy (AFM) (1). In this study, we analyze the molecular organization of these striated domains using AFM, photo-cross-linking, fluorescence spectroscopy, nuclear magnetic resonance (NMR), and X-ray diffraction techniques on different functionalized TM peptides. The results demonstrate that the striated domains consist of linear arrays of single TM peptides with a dominantly antiparallel organization in which the peptides interact with each other and with lipids. The peptide arrays are regularly spaced by +/-8.5 nm and are separated by somewhat perturbed gel-state lipids with hexagonally organized acyl chains, which have lost their tilt. This system provides an example of how domains of peptides and lipids can be formed in membranes as a result of a combination of specific peptide-peptide and peptide-lipid interactions.     

Cryo-electron microscopy of artificial biological membranes

Vitrified synthetic phosphatidycholine liposome suspensions were studied by cryo-electron microscopy. The bilayer structure is resolved on vitrified liposome images. The packing of the aliphatic chains of the lipid within vitrified liposomes can be determined by the analysis of electron diffraction patterns. Images and electron diffraction patterns show that the structure of vitrified liposomes is related to the structure that liposomes have before vitrification. In fact, vitrified liposomes have a different structure, depending whether they are maintained before cooling at a temperature higher or lower than that corresponding to the ‘melting’ of the hydrocarbon chain of the lipids. Below the melting temperature, liposomes are formed by small domains.     

Glycosylation induces shifts in the lateral distribution of cholesterol from ordered towards less ordered domains

Several studies have indicated the involvement of steryl glycosides in the cellular stress response. In this work, we have compared the effect of 1-O-cholesteryl-β-d-glucoside, 1-O-cholesteryl-β-d-galactoside and cholesterol on the properties of glycerophospholipid and sphingolipid bilayers. The studies were performed in order to gain insight into the change in membrane properties that would follow upon the glycosylation of cholesterol in cells subjected to stress. DPH anisotropy measurements indicated that the cholesteryl glycosides (10-40 mol%) increased the order of the hydrophobic region of a POPC bilayer almost as efficiently as cholesterol. In a PSM bilayer, the cholesteryl glycosides were however shown to be much less effective compared to cholesterol in ordering the hydrocarbon chain region at temperatures above the gel to liquid-crystalline phase transition. Fluorescence quenching analysis of multicomponent lipid bilayers demonstrated that the cholesteryl glycosides, in contrast to cholesterol, were unable to stabilize ordered domains rich in PSM against temperature-induced dissociation. When the sterols were incorporated into bilayers composed of both POPC and PSM, the cholesteryl glycosides showed a higher propensity, compared to cholesterol, to influence the endothermal component representing the melting of POPC-rich domains, as determined by differential scanning calorimetry. Taken together, the results indicate that the glycosylation of cholesterol diminishes the ability of the sterol to reside in lateral domains constituted by membrane lipids having highly ordered hydrocarbon chains.     

Lipid-binding proteins as stabilizers of membrane microdomains--possible physiological significance

Below the melting point temperature of lipids, artificial lipid membranes usually exist in the ordered gel phase. Above these temperatures lipid acyl chains become fluid and disordered (liquid-crystalline phase). Depending on the chemical composition of artificial membranes, phase separation may occur, leading to the formation of transient or stable membrane domains. A similar phase separation of lipids into ordered and disordered domains has been observed in natural membranes at physiological temperature range. Moreover, it has been reported that certain proteins prefer certain organization of lipids, as for example glycosylphosphatidylinositol-anchored proteins or Src family of tyrosine kinases. The aim of present review is to discuss the possibility that some lipid microdomains are induced or stabilized by lipid-binding proteins that under certain conditions, for example due to a rise of cytosolic Ca2+ or pH changes, may attach to the membrane surface, inducing clustering of lipid molecules and creation of ordered lipid microdomains. These domains may than attract other cytosolic proteins, either enzymes or regulatory proteins. It is, therefore, postulated that lipid microdomains play important roles within a cell, in signal transduction and enzymatic catalysis, and also in various pathological states, as Alzheimer's disease, anti-phosphatidylserine syndrome, or development of multidrug resistance of cancer cells.     

Studies of highly asymmetric mixed-chain diacyl phosphatidylcholines that form mixed-interdigitated gel phases: Fourier transform infrared and 2H NMR spectroscopic studies of hydrocarbon chain conformation and orientational order in the liquid-crystalline state.

Hydrocarbon chain conformational and orientational order in liquid-crystalline bilayers of the highly chain-asymmetric 1-O-eicosanoyl, 2-O-dodecanoyl and 1-O-decanoyl, 2-O-docosanoyl phosphatidylcholines were studied by Fourier transform infrared (FTIR) and deuterium nuclear magnetic resonance (2H-NMR) spectroscopy, respectively, and compared with appropriate symmetric-chain phosphatidylcholines at comparable reduced temperatures. FTIR spectroscopy indicates that these two asymmetric-chain phospholipids contain a slightly greater number of kink, a considerably larger number of double-gauche, but a somewhat smaller number of end-gauche conformers than does dipalmitoylphosphatidylcholine, a symmetric-chain phospholipid having the same total number of carbon atoms in its hydrocarbon chains. Moreover, the asymmetric-chain phospholipids also contain a larger total number of gauche conformers, suggesting that their hydrocarbon chains are more disordered overall than are those of dipalmitoylphosphatidylcholine. 2H-NMR studies of the specifically chain-perdeuterated analogs of these asymmetric-chain lipids reveal that the orientational order parameter profiles of their shorter and longer chains differ both qualitatively and quantitatively, regardless of whether they are esterified at the sn1- or sn2 positions of the glycerol molecule. The longer hydrocarbon chains exhibit unusual orientational order profiles in which the order gradient is steepest in the middle of the chain and relatively shallower in regions adjacent to the carboxyl and methyl termini, whereas the short hydrocarbon chains exhibit orientational order profiles typical of those commonly observed with conventional symmetric chain lipids. When compared at equivalent depths in the bilayer, the shorter hydrocarbon chains of the asymmetric-chain lipids are more orientationally disordered than are their longer chain counterparts. At comparable reduced temperatures, the shorter and longer chains of the asymmetric-chain lipids are more orientationally disordered than those of appropriate short and long symmetric-chain lipids, but the chain-averaged orientational order of the symmetric-chain lipid decreases more sharply with increases in temperature than does that of the comparable chain of the asymmetric-chain species. Moreover, the order plateau regions adjacent to the carboxyl groups of the longer chains of the asymmetric-chain phosphatidylcholines are shorter than those of symmetric-chain lipids of comparable hydrocarbon chain length. Overall, the data indicate that the conformational and orientational order in the liquid-crystalline states of these highly asymmetric-chain lipids differ significantly from those of comparable symmetric-chain lipids. Also, the unusual shape of the orientational order profile of the longer chains of the former is attributed to interaction between the methyl termini regions of the long chains with hydrocarbon chains in opposing monolayers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ceramide selectively displaces cholesterol from ordered lipid domains (rafts): implications for lipid raft structure and function

Ceramide is a membrane lipid involved in a number of crucial biological processes. Recent evidence suggests that ceramide is likely to reside and function within lipid rafts; ordered sphingolipid and cholesterol-rich lipid domains believed to exist within many eukaryotic cell membranes. Using lipid vesicles containing co-existing raft domains and disordered fluid domains, we find that natural and saturated synthetic ceramides displace sterols from rafts. Other raft lipids remain raft-associated in the presence of ceramide, showing displacement is relatively specific for sterols. Like cholesterol-containing rafts, ceramide-rich "rafts" remain in a highly ordered state. Comparison of the sterol-displacing abilities of natural ceramides with those of saturated diglycerides and an unsaturated ceramide demonstrates that tight lipid packing is critical for sterol displacement by ceramide. Based on these results, and the fact that cholesterol and ceramides both have small polar headgroups, we propose that ceramides and cholesterol compete for association with rafts because of a limited capacity of raft lipids with large headgroups to accommodate small headgroup lipids in a manner that prevents unfavorable contact between the hydrocarbon groups of the small headgroup lipids and the surrounding aqueous environment. Minimizing the exposure of cholesterol and ceramide to water may be a strong driving force for the association of other molecules with rafts. Furthermore, displacement of sterol from rafts by ceramide is very likely to have marked effects upon raft structure and function, altering liquid ordered properties as well as molecular composition. In this regard, certain previously observed physiological processes may be a result of displacement. In particular, a direct connection to the previously observed sphingomyelinase-induced displacement of cholesterol from plasma membranes in cells is proposed.     

Biophysical properties of membrane lipids of anammox bacteria: I. Ladderane phospholipids form highly organized fluid membranes

Anammox bacteria that are capable of anaerobically oxidizing ammonium (anammox) with nitrite to nitrogen gas produce unique membrane phospholipids that comprise hydrocarbon chains with three or five linearly condensed cyclobutane rings. To gain insight into the biophysical properties of these ‘ladderane’ lipids, we have isolated a ladderane phosphatidylcholine and a mixed ladderane phosphatidylethanolamine/phosphatidylglycerol lipid fraction and reconstituted these lipids in different membrane environments. Langmuir monolayer experiments demonstrated that the purified ladderane phospholipids form fluid films with a relatively high lipid packing density. Fluid-like behavior was also observed for ladderane lipids in bilayer systems as monitored by cryo-electron microscopy on large unilamellar vesicles (LUVs) and epi-fluorescence microscopy on giant unilamellar vesicles (GUVs). Analysis of the LUVs by fluorescence depolarization revealed a relatively high acyl chain ordering in the hydrophobic region of the ladderane phospholipids. Micropipette aspiration experiments were applied to study the mechanical properties of ladderane containing lipid bilayers and showed a relatively high apparent area compressibility modulus for ladderane containing GUVs, thereby confirming the fluid and acyl chain ordered characteristics of these lipids. The biophysical findings in this study support the previous postulation that dense membranes in anammox cells protect these microbes against the highly toxic and volatile anammox metabolites.