Effective Attraction Interactions between Like-charge Macroions Bound to Binary Fluid Lipid Membranes

Using integral equation theory of liquids to a binary mixed fluid lipid membrane, we study the membrane-mediated interactions between the macroions and the redistribution of neutral and charged lipids due to binding macroions. We find that when the concentration of binding macroions is infinitely dilute, the main contribution to the attractive potential between macroions is the line tension between neutral and charged lipids of the membrane, and the bridging effect also contributes to the attraction. As the relative concentration of charged lipids is increased, we observe a repulsive - attractive - repulsive potential transition due to the competition between the line tension of lipids and screened electrostatic macroion-macroion interactions. For the finite concentration of macroions, the main feature of the attraction is similar to the infinite dilution case. However, due to the interplay of formation of charged lipid - macroion complexes, the line tension of redistributed binary lipids induced by single macroion is lowered in this case, and the maximum of attractive potential will shift toward the higher values of the charged lipid concentration

Coupling between pore formation and phase separation in charged lipid membranes

We investigated the effect of charge on the membrane morphology of giant unilamellar vesicles (GUVs) composed of various mixtures containing charged lipids. We observed the membrane morphologies by fluorescent and confocal laser microscopy in lipid mixtures consisting of a neutral unsaturated lipid [dioleoylphosphatidylcholine (DOPC)], a neutral saturated lipid [dipalmitoylphosphatidylcholine (DPPC)], a charged unsaturated lipid [dioleoylphosphatidylglycerol (DOPG$^{\scriptsize{(-)}}$)], a charged saturated lipid [dipalmitoylphosphatidylglycerol (DPPG$^{\scriptsize{(-)}}$)], and cholesterol (Chol). In binary mixtures of neutral DOPC/DPPC and charged DOPC/DPPG$^{\scriptsize{(-)}}$, spherical vesicles were formed. On the other hand, pore formation was often observed with GUVs consisting of DOPG$^{\scriptsize{(-)}}$ and DPPC. In a DPPC/DPPG$^{\scriptsize{(-)}}$/Chol ternary mixture, pore-formed vesicles were also frequently observed. The percentage of pore-formed vesicles increased with the DPPG$^{\scriptsize{(-)}}$ concentration. Moreover, when the head group charges of charged lipids were screened by the addition of salt, pore-formed vesicles were suppressed in both the binary and ternary charged lipid mixtures. We discuss the mechanisms of pore formation in charged lipid mixtures and the relationship between phase separation and the membrane morphology. Finally, we reproduce the results seen in experimental systems by using coarse-grained molecular dynamics simulations.Comment: 34 pages, 10 figure

Charge-induced phase separation in lipid membranes

The phase separation in lipid bilayers that include negatively charged lipids is examined experimentally. We observed phase-separated structures and determined the membrane miscibility temperatures in several binary and ternary lipid mixtures of unsaturated neutral lipid, dioleoylphosphatidylcholine (DOPC), saturated neutral lipid, dipalmitoylphosphatidylcholine (DPPC), unsaturated charged lipid, dioleoylphosphatidylglycerol (DOPG$^{\scriptsize{(-)}}$), saturated charged lipid, dipalmitoylphosphatidylglycerol (DPPG$^{\scriptsize{(-)}}$), and cholesterol. In binary mixtures of saturated and unsaturated charged lipids, the combination of the charged head with the saturation of hydrocarbon tail is a dominant factor for the stability of membrane phase separation. DPPG$^{\scriptsize{(-)}}$ enhances phase separation, while DOPG$^{\scriptsize{(-)}}$ suppresses it. Furthermore, the addition of DPPG$^{\scriptsize{(-)}}$ to a binary mixture of DPPC/cholesterol induces phase separation between DPPG$^{\scriptsize{(-)}}$-rich and cholesterol-rich phases. This indicates that cholesterol localization depends strongly on the electric charge on the hydrophilic head group rather than on the ordering of the hydrocarbon tails. Finally, when DPPG$^{\scriptsize{(-)}}$ was added to a neutral ternary system of DOPC/DPPC/Cholesterol (a conventional model of membrane rafts), a three-phase coexistence was produced. We conclude by discussing some qualitative features of the phase behaviour in charged membranes using a free energy approach.Comment: 17 pages, 6 figure

A method for microdetermination of major neutral lipids and an application of the procedure to tissue lipids

Cholesterol, cholesteryl esters, triglycerides and fatty acids as major neutral lipids and phospholipids were examined in quantitative analysis. The method consisted of three steps: (1) separation of lipids by one-dimensional thin-layer chromatography on silica gel plates; (2) elution of neutral lipids from scraped silica gel with chloroform-methanol (4:1); and (3) colorimetric determination of individual neutral lipids in eluates and phospholipids in silica gel. The conditions were modified for chromotropic acid reaction for determining triglycerides. Laurell's method for determining fatty acids was also modified to apply to quantitative thin-layer chromatography. The accuracy of the modified methods was well-defined as the absorbance values were on a linear curve. A quantitative study was made of the recovery of triglycerides and fatty acids after chromatography. Combining these modified methods and colorimetry for determination of cholesterol cholesteryl esters and phospholipids, the author established a micromethod for determining the major neutral lipids and phospholipids by thin-layer chromatography. Lipids from HeLa, S-3 cells were analyzed to examine the applicability of this method to tissues. The results indicated that the new method permitted a reliable estimation of the major neutral lipids and phospholipids from small amounts of tissues.</p

ER stress in antigen‐presenting cells promotes NKT cell activation through endogenous neutral lipids

CD1d-restricted invariant natural killer T (iNKT) cells constitute a common glycolipid-reactive innate-like T-cell subset with a broad impact on innate and adaptive immunity. While several microbial glycolipids are known to activate iNKT cells, the cellular mechanisms leading to endogenous CD1d-dependent glycolipid responses remain largely unclear. Here, we show that endoplasmic reticulum (ER) stress in APCs is a potent inducer of CD1d-dependent iNKT cell autoreactivity. This pathway relies on the presence of two transducers of the unfolded protein response: inositol-requiring enzyme-1a (IRE1α) and protein kinase R-like ER kinase (PERK). Surprisingly, the neutral but not the polar lipids generated within APCs undergoing ER stress are capable of activating iNKT cells. These data reveal that ER stress is an important mechanism to elicit endogenous CD1d-restricted iNKT cell responses through induction of distinct classes of neutral lipids

Acute Hypercapnia/Ischemia Alters the Esterification of Arachidonic Acid and Docosahexaenoic Acid Epoxide Metabolites in Rat Brain Neutral Lipids.

In the brain, approximately 90% of oxylipins are esterified to lipids. However, the significance of this esterification process is not known. In the present study, we (1) validated an aminopropyl solid phase extraction (SPE) method for separating esterified lipids using 100 and 500 mg columns and (2) applied the method to quantify the distribution of esterified oxylipins within phospholipids (PL) and neutral lipids (NL) (i.e. triacylglycerol and cholesteryl ester) in rats subjected to head-focused microwave fixation (controls) or CO2 -induced hypercapnia/ischemia. We hypothesized that oxylipin esterification into these lipid pools will be altered following CO2 -induced hypercapnia/ischemia. Lipids were extracted from control (n = 8) and CO2 -asphyxiated (n = 8) rat brains and separated on aminopropyl cartridges to yield PL and NL. The separated lipid fractions were hydrolyzed, purified with hydrophobic-lipophilic-balanced SPE columns, and analyzed with ultra-high-pressure liquid chromatography coupled to tandem mass spectrometry. Method validation showed that the 500 mg (vs 100 mg) aminopropyl columns yielded acceptable separation and recovery of esterified fatty acid epoxides but not other oxylipins. Two epoxides of arachidonic acid (ARA) were significantly increased, and three epoxides of docosahexaenoic acid (DHA) were significantly decreased in brain NL of CO2 -asphyxiated rats compared to controls subjected to head-focused microwave fixation. PL-bound fatty acid epoxides were highly variable and did not differ significantly between the groups. This study demonstrates that hypercapnia/ischemia alters the concentration of ARA and DHA epoxides within NL, reflecting an active turnover process regulating brain fatty acid epoxide concentrations

Effect of charged lipids on the ionization behavior of glutamic acid containing transmembrane helices

Transmembrane proteins make up critical components of living cells. Protein function can be greatly impacted by the charged state of its respective components, the side chains of amino acid residues. Thus far, in the lipid membrane, little is known about the properties of residues such as glutamic acid. To explore these properties, I have included glutamic acid in a suitable model peptide-lipid system for fundamental biophysical experiments. Within the system, I have placed a glutamic acid residue instead of leucine in the L14 position of the helical hydrophobic peptide GWALP23 (acetyl-GGALWLALALALAL14ALALWLAGA-amide). Substitutions of glutamine and aspartic acid serve as controls for the properties of the peptide helix in lipid bilayer membranes. The GWALP23 peptide derivatives are placed in various lipid bilayer environments. Specifically, I investigated the impact of glutamic acid (position E14) when differently charged lipids are present in the bilayer. The underlying importance is to understand the charged or neutral state behavior of glutamic acid under conditions where it is important for the functioning of several types of membrane proteins, such as ion channels, drug transporters and others. For the experimental plan, core alanine resides of GWALP23 were labeled with deuterium to enable detection of helix characteristics by solid-state 2H NMR spectroscopy. The peptide-lipid samples included primarily the neutral lipid DMPC, 1,2-dimyristoylphosphatidylcholine, (with 14-carbon acyl chains), along with 10% of a charged lipid. For each membrane system, I confirmed lipid bilayer formation for the particular peptide-lipid mixture by solid-state 31P NMR. The charged lipids consisted of the negatively charged lipid DMPG, 1,2-dimyristoylphosphatidylglycerol, and the positively charged lipid DMTAP, 1,2-dimyristoyl-3-trimethylammonium-propane. These charged lipids were found to influence the properties of the GWALP23 helix when E14 was present. DMTAP, in particular, improves the 2H NMR spectra and the prospects for characterizing helix dynamics when a glutamic acid residue is present. While some experiments were cut short due to a global emergency, the results show promise for characterizing glutamic acid in model helices and actual membrane proteins

Coarse-grained molecular dynamics simulation of binary charged lipid membranes: Phase separation and morphological dynamics

Biomembranes, which are mainly composed of neutral and charged lipids, exhibit a large variety of functional structures and dynamics. Here, we report a coarse-grained molecular dynamics (MD) simulation of the phase separation and morphological dynamics in charged lipid bilayer vesicles. The screened long-range electrostatic repulsion among charged head groups delays or inhibits the lateral phase separation in charged vesicles compared with neutral vesicles, suggesting the transition of the phase-separation mechanism from spinodal decomposition to nucleation or homogeneous dispersion. Moreover, the electrostatic repulsion causes morphological changes, such as pore formation, and further transformations into disk, string, and bicelle structures, which are spatiotemporally coupled to the lateral segregation of charged lipids. Based on our coarse-grained MD simulation, we propose a plausible mechanism of pore formation at the molecular level. The pore formation in a charged-lipid-rich domain is initiated by the prior disturbance of the local molecular orientation in the domain.Comment: 12pages, 9 figure