Activation of hTREK-1 by polyunsaturated fatty acids involves direct interaction.

TREK-1 is a mechanosensitive channel activated by polyunsaturated fatty acids (PUFAs). Its activation is supposed to be linked to changes in membrane tension following PUFAs insertion. Here, we compared the effect of 11 fatty acids and ML402 on TREK-1 channel activation using the whole cell and the inside-out configurations of the patch-clamp technique. Firstly, TREK-1 activation by PUFAs is variable and related to the variable constitutive activity of TREK-1. We observed no correlation between TREK-1 activation and acyl chain length or number of double bonds suggesting that the bilayer-couple hypothesis cannot explain by itself the activation of TREK-1 by PUFAs. The membrane fluidity measurement is not modified by PUFAs at 10 µM. The spectral shift analysis in TREK-1-enriched microsomes indicates a KD,TREK1 at 44 µM of C22:6 n-3. PUFAs display the same activation and reversible kinetics than the direct activator ML402 and activate TREK-1 in both whole-cell and inside-out configurations of patch-clamp suggesting that the binding site of PUFAs is accessible from both sides of the membrane, as for ML402. Finally, we proposed a two steps mechanism: first, insertion into the membrane, with no fluidity or curvature modifications at 10 µM, and then interaction with TREK-1 channel to open it.

Acrolein Induces Changes in Cell Membrane and Cytosol Proteins of Erythrocytes.

High concentrations of acrolein (2-propenal) are found in polluted air and cigarette smoke, and may also be generated endogenously. Acrolein is also associated with the induction and progression of many diseases. The high reactivity of acrolein towards the thiol and amino groups of amino acids may cause damage to cell proteins. Acrolein may be responsible for the induction of oxidative stress in cells. We hypothesized that acrolein may contribute to the protein damage in erythrocytes, leading to the disruption of the structure of cell membranes. The lipid membrane fluidity, membrane cytoskeleton, and osmotic fragility were measured for erythrocytes incubated with acrolein for 24 h. The levels of thiol, amino, and carbonyl groups were determined in cell membrane and cytosol proteins. The level of non-enzymatic antioxidant potential (NEAC) and TBARS was also measured. The obtained research results showed that the exposure of erythrocytes to acrolein causes changes in the cell membrane and cytosol proteins. Acrolein stiffens the cell membrane of erythrocytes and increases their osmotic sensitivity. Moreover, it has been shown that erythrocytes treated with acrolein significantly reduce the non-enzymatic antioxidant potential of the cytosol compared to the control.

Defective pgsA contributes to increased membrane fluidity and cell wall thickening in Staphylococcus aureus with high-level daptomycin resistance.

Daptomycin is a membrane-targeting last-resort antimicrobial therapeutic for the treatment of infections caused by methicillin- and/or vancomycin-resistant Staphylococcus aureus. In the rare event of failed daptomycin therapy, the source of resistance is often attributable to mutations directly within the membrane phospholipid biosynthetic pathway of S. aureus or in the regulatory systems that control cell envelope response and membrane homeostasis. Here we describe the structural changes to the cell envelope in a daptomycin-resistant isolate of S. aureus strain N315 that has acquired mutations in the genes most commonly reported associated with daptomycin resistance: mprF, yycG, and pgsA. In addition to the decreased phosphatidylglycerol (PG) levels that are the hallmark of daptomycin resistance, the mutant with high-level daptomycin resistance had increased branched-chain fatty acids (BCFAs) in its membrane lipids, increased membrane fluidity, and increased cell wall thickness. However, the successful utilization of isotope-labeled straight-chain fatty acids (SCFAs) in lipid synthesis suggested that the aberrant BCFA:SCFA ratio arose from upstream alteration in fatty acid synthesis rather than a structural preference in PgsA. Transcriptomics studies revealed that expression of pyruvate dehydrogenase (pdhB) was suppressed in the daptomycin-resistant isolate, which is known to increase BCFA levels. While complementation with an additional copy of pdhB had no effect, complementation of the pgsA mutation resulted in increased PG formation, reduction in cell wall thickness, restoration of normal BCFA levels, and increased daptomycin susceptibility. Collectively, these results demonstrate that pgsA contributes to daptomycin resistance through its influence on membrane fluidity and cell wall thickness, in addition to phosphatidylglycerol levels. IMPORTANCE: The cationic lipopeptide antimicrobial daptomycin has become an essential tool for combating infections with Staphylococcus aureus that display reduced susceptibility to ß-lactams or vancomycin. Since daptomycin's activity is based on interaction with the negatively charged membrane of S. aureus, routes to daptomycin-resistance occur through mutations in the lipid biosynthetic pathway surrounding phosphatidylglycerols and the regulatory systems that control cell envelope homeostasis. Therefore, there are many avenues to achieve daptomycin resistance and several different, and sometimes contradictory, phenotypes of daptomycin-resistant S. aureus, including both increased and decreased cell wall thickness and membrane fluidity. This study is significant because it demonstrates the unexpected influence of a lipid biosynthesis gene, pgsA, on membrane fluidity and cell wall thickness in S. aureus with high-level daptomycin resistance.

Fatty acid composition and biophysical characteristics of the cell membrane of feline spermatozoa.

Sperm membrane composition and biophysical characteristics play a pivotal role in many physiological processes (i.e. sperm motility, capacitation, acrosome reaction and fusion with the oocyte) as well as in semen processing (e.g. cryopreservation). The aim of this study was to characterize the fatty acid content and biophysical characteristics (anisotropy, generalized polarization) of the cell membrane of domestic cat spermatozoa. Semen was collected from 34 adult male cats by urethral catheterization. After a basic semen evaluation, the fatty acid content of some of the samples (n = 11) was evaluated by gas chromatography. Samples from other individuals (n = 23) were subjected to biophysical analysis: membrane anisotropy (which is inversely proportional to membrane fluidity) and generalized polarization (describing lipid order); both measured by fluorimetry at three temperature points: 38 °C, 25 °C and 5 °C. Spermatozoa from some samples (n = 10) were cryopreserved in TRIS egg yolk-glycerol extender and underwent the same biophysical analysis after thawing. Most fatty acids in feline spermatozoa were saturated (69.76 ± 24.45%), whereas the polyunsaturated fatty acid (PUFA) content was relatively low (6.12 ± 5.80%). Lowering the temperature caused a significant decrease in membrane fluidity and an increase in generalized polarization in fresh spermatozoa, and these effects were even more pronounced following cryopreservation. Anisotropy at 38 °C in fresh samples showed strong positive correlations with viability and motility parameters after thawing. In summary, feline spermatozoa are characterized by a very low PUFA content and a low ratio of unsaturated:saturated fatty acids, which may contribute to low oxidative stress. Cryopreservation alters the structure of the sperm membrane, increasing the fluidity of the hydrophobic portion of the bilayer and the lipid order in the hydrophilic portion. Because lower membrane fluidity in fresh semen was linked with better viability and motility after cryopreservation, this parameter may be considered an important factor in determination of sperm cryoresistance.

Triplet-Triplet Annihilation Upconverting Liposomes: Mechanistic Insights into the Role of Membranes in Two-Dimensional TTA-UC.

Triplet-triplet annihilation upconversion (TTA-UC) implemented in nanoparticle assemblies is of emerging interest in biomedical applications, including in drug delivery and imaging. As it is a bimolecular process, ensuring sufficient mobility of the sensitizer and annihilator to facilitate effective collision in the nanoparticle is key. Liposomes can provide the benefits of two-dimensional confinement and condensed concentration of the sensitizer and annihilator along with superior fluidity compared to other nanoparticle assemblies. They are also biocompatible and widely applied across drug delivery modalities. However, there are relatively few liposomal TTA-UC systems reported to date, so systematic studies of the influence of the liposomal environment on TTA-UC are currently lacking. Here, we report the first example of a BODIPY-based sensitizer TTA-UC system within liposomes and use this system to study TTA-UC generation and compare the relative intensity of the anti-Stokes signal for this system as a function of liposome composition and membrane fluidity. We report for the first time on time-resolved spectroscopic studies of TTA-UC in membranes. Nanosecond transient absorption data reveal the BODIPY-perylene dyad sensitizer has a long triplet lifetime in liposome with contributions from three triplet excited states, whose lifetimes are reduced upon coinclusion of the annihilator due to triplet-triplet energy transfer, to a greater extent than in solution. This indicates triplet energy transfer between the sensitizer and the annihilator is enhanced in the membrane system. Molecular dynamics simulations of the sensitizer and annihilator TTA collision complex are modeled in the membrane and confirm the co-orientation of the pair within the membrane structure and that the persistence time of the bound complex exceeds the TTA kinetics. Modeling also reliably predicted the diffusion coefficient for the sensitizer which matches closely with the experimental values from fluorescence correlation spectroscopy. The relative intensity of the TTA-UC output across nine liposomal systems of different lipid compositions was explored to examine the influence of membrane viscosity on upconversion (UC). UC showed the highest relative intensity for the most fluidic membranes and the weakest intensity for highly viscous membrane compositions, including a phase separation membrane. Overall, our study reveals that the co-orientation of the UC pair within the membrane is crucial for effective TTA-UC within a biomembrane and that the intensity of the TTA-UC output can be tuned in liposomal nanoparticles by modifying the phase and fluidity of the liposome. These new insights will aid in the design of liposomal TTA-UC systems for biomedical applications.

Lipid phase separation impairs membrane thickness sensing by the Bacillus subtilis sensor kinase DesK.

Membrane fluidity and thickness have emerged as crucial factors for the activity of and resistance to several antimicrobials. However, the lack of tools to study membrane fluidity and, in particular, thickness in living bacteria limits our understanding of this interplay. The Bacillus subtilis histidine kinase/phosphatase DesK is a molecular sensor that directly detects membrane thickness. It controls activity of DesR, which regulates expression of the lipid desaturase Des, known for its role in cold adaptation and daptomycin susceptibility. We hypothesized that this property could be exploited to develop biosensors and reporters for antibiotic-induced changes in membrane fluidity and thickness. To test this, we designed three assays based on the des system: activation of the Pdes promoter as reporter for membrane thickening, localization of DesK-GFP(green-fluorescent protein) as proxy for rigidified membrane domains, and antibiotic sensitivity of des, desK, and desR deletion mutants as readout for the importance of membrane rigidification/thickening under the tested condition. While we could not confirm the suitability of the des system as reporter for antibiotic-induced changes in membrane thickness, we did observe that des expression is only activated by mild temperature shocks, likely due to partitioning of the sensor DesK into fluid membrane domains upon phase separation, precluding effective thickness sensing under harsh cold shock and antibiotic stress conditions. Similarly, we did not observe any sensitivity of the deletion mutants to either temperature or antibiotic stress, raising the question to what extent the des system contributes to fluidity adaptation under these conditions. IMPORTANCE: The B. subtilis des system is a prime model for direct molecular membrane thickness sensor and, as such, has been well studied in vitro. Our study shows that our understanding of its function in vivo and its importance under temperature and antibiotic stress is still very limited. Specifically, our results suggest that (i) the des system senses very subtle membrane fluidity changes that escape detection by established fluidity reporters like laurdan; (ii) membrane thickness sensing by DesK is impaired by phase separation due to partitioning of the protein into the fluid phase; and (iii) fluidity adaptations by Des are too subtle to elicit growth defects under rigidifying conditions, raising the question of how much the des system contributes to adaptation of overall membrane fluidity.

Protein-Mediated Changes in Membrane Fluidity and Ordering: Insights into the Molecular Mechanism and Implications for Cellular Function.

Probing protein-membrane interactions is vital for understanding biological functionality for various applications such as drug development, targeted drug delivery, and creation of functional biomaterials for medical and industrial purposes. In this study, we have investigated interaction of Human Serum Albumin (HSA) with two different lipids, dipalmitoylphosphatidylglycerol (dDPPG) and dipalmitoylphosphatidylcholine (dDPPC), using Vibrational Sum Frequency Generation spectroscopy at different membrane fluidity values. In the liquid-expanded (LE) state of the lipid, HSA (at pH 3.5) deeply intercalated lipid chains through a combination of electrostatic and hydrophobic interactions, which resulted in more ordering of the lipid chains. However, in the liquid-condensed (LC) state, protein intercalation is decreased due to tighter lipid packing. Moreover, our findings revealed distinct differences in HSA's interaction with dDPPG and dDPPC lipids. The interaction with dDPPC remained relatively weak compared to dDPPG. These results shed light on the significance of protein mediated changes in lipid characteristics, which hold considerable implications for understanding membrane protein behavior, lipid-mediated cellular processes, and lipid-based biomaterial design.

Membrane fluidity properties of lipid-coated polylactic acid nanoparticles.

Lipid coating is considered a versatile strategy to equip nanoparticles (NPs) with a biomimetic surface coating, but the membrane properties of these nanoassemblies remain in many cases insufficiently understood. In this work, we apply C-Laurdan generalized polarization (GP) measurements to probe the temperature-dependent polarity of hybrid membranes consisting of a lipid monolayer adsorbed onto a polylactic acid (PLA) polymer core as function of lipid composition and compare the behavior of the lipid coated NPs (LNPs) with that of liposomes assembled from identical lipid mixtures. The LNPs were generated by nanoprecipitation of the polymer in aqueous solutions containing two types of lipid mixtures: (i) cholesterol, dipalmitoylphosphatidylcholine (DPPC), and the ganglioside GM3, as well as (ii) dioleoylphosphatidylcholine (DOPC), DPPC and GM3. LNPs were found to exhibit more distinct and narrower phase transitions than corresponding liposomes and to retain detectable phase transitions even for cholesterol or DOPC concentrations that yielded no detectable transitions in liposomes. These findings together with higher GP values in the case of the LNPs for temperatures above the phase transition temperature indicate a stabilization of the membrane through the polymer core. LNP binding studies to GM3-recognizing cells indicate that differences in the membrane fluidity affect binding avidity in the investigated model system.

St. John's wort extract Ze 117 alters the membrane fluidity of C6 glioma cells by influencing cellular cholesterol metabolism.

Chronic stress is associated with major depressive disorder (MDD). Increased glucocorticoid levels caused by uncontrolled release through the hypothalamic‒pituitary‒adrenal (HPA) axis can cause changes in the lipid content of the cellular plasma membrane. These changes are suspected to be involved in the development of depressive disorders. St. John's wort extract (SJW) Ze 117 has long been used as an alternative to synthetic antidepressants. Part of its effect may be due to an effect on the cellular lipid composition and thus on the properties of plasma membranes and receptor systems embedded therein. In this study, we investigated the effect of Ze 117 on that of dexamethasone and simvastatin. Dexamethasone increases the fluidity of C6 cell plasma membranes. This effect is counteracted by administration of Ze 117. Here we demonstrate that this is not due to a change in C16:1/16:0 and C18:1/18:0 ratios in C6 cell fatty acids. On the other hand, Ze 117 increased the cellular cholesterol content by 42.5%, whereas dexamethasone reduced cholesterol levels similarly to simvastatin. Lowering cholesterol levels by dexamethasone or simvastatin resulted in decreased ß-arrestin 2 recruitment to the 5-HT1a receptor. This effect was counterbalanced by Ze 117, whereas the SJW extract had little effect on ß-arrestin 2 recruitment in non-stressed cells. Taken together, in C6 cells, Ze 117 induces changes in membrane fluidity through its effect on cellular cholesterol metabolism rather than by affecting fatty acid saturation. This effect is reflected in an altered signal transduction of the 5-HT1a receptor under Ze 117 administration. The current in vitro results support the hypothesis that Ze 117 addresses relevant parts of the cellular lipid metabolism, possibly explaining some of the antidepressant actions of Ze 117.

S-Adenosyl-l-methionine restores brain mitochondrial membrane fluidity and GSH content improving Niemann-Pick type C disease.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by impaired motor coordination due to neurological defects and cerebellar dysfunction caused by the accumulation of cholesterol in endolysosomes. Besides the increase in lysosomal cholesterol, mitochondria are also enriched in cholesterol, which leads to decreased membrane fluidity, impaired mitochondrial function and loss of GSH, and has been shown to contribute to the progression of NPC disease. S-Adenosyl-l-methionine (SAM) regulates membrane physical properties through the generation of phosphatidylcholine (PC) from phosphatidylethanolamine (PE) methylation and functions as a GSH precursor by providing cysteine in the transsulfuration pathway. However, the role of SAM in NPC disease has not been investigated. Here we report that Npc1-/- mice exhibit decreased brain SAM levels but unchanged S-adenosyl-l-homocysteine content and lower expression of Mat2a. Brain mitochondria from Npc1-/- mice display decreased mitochondrial GSH levels and liquid chromatography-high resolution mass spectrometry analysis reveal a lower PC/PE ratio in mitochondria, contributing to increased mitochondrial membrane order. In vivo treatment of Npc1-/- mice with SAM restores SAM levels in mitochondria, resulting in increased PC/PE ratio, mitochondrial membrane fluidity and subsequent replenishment of mitochondrial GSH levels. In vivo SAM treatment improves the decline of locomotor activity, increases Purkinje cell survival in the cerebellum and extends the average and maximal life spam of Npc1-/- mice. These findings identify SAM as a potential therapeutic approach for the treatment of NPC disease.

Erythrocytes membrane fluidity changes induced by adenylyl cyclase cascade activation: study using fluorescence recovery after photobleaching.

In this study, fluorescence recovery after photobleaching (FRAP) experiments were performed on RBC labeled by lipophilic fluorescent dye CM-DiI to evaluate the role of adenylyl cyclase cascade activation in changes of lateral diffusion of erythrocytes membrane lipids. Stimulation of adrenergic receptors with epinephrine (adrenaline) or metaproterenol led to the significant acceleration of the FRAP recovery, thus indicating an elevated membrane fluidity. The effect of the stimulation of protein kinase A with membrane-permeable analog of cAMP followed the same trend but was less significant. The observed effects are assumed to be driven by increased mobility of phospholipids resulting from the weakened interaction between the intermembrane proteins and RBC cytoskeleton due to activation of adenylyl cyclase signaling cascade.

Effect of citric acid on cell membrane structure and function of Issatchenkia terricola WJL-G4.

AIMS: This study aimed to investigate the changes of cell membrane structure and function of Issatchenkia terricola under citric acid by performing physiological analysis. METHODS AND RESULTS: The membrane integrity, surface hydrophobicity, structure, fluidity, apoptosis, and fatty acid methyl esters composition of I. terricola WJL-G4 cells were determined by propidium iodide staining, microbial adhesion to hydrocarbon test, transmission electron microscopy analysis, fluorescence anisotropy, flow cytometry, and gas chromatography-mass, respectively. The results showed that with the increasing of citric acid concentrations, the cell vitality, membrane integrity, and fluidity of I. terricola reduced; meanwhile, apoptosis rate, membrane permeable, hydrophobicity, and ergosterol contents augmented significantly. Compared to control, the activities of Na+, K+-ATPase, and Ca2+, Mg2+-ATPase increased by 3.73-fold and 6.70-fold, respectively, when citric acid concentration increased to 20 g l-1. The cells cracked and their cytoplasm effused when the citric acid concentration reached 80 g l-1. CONCLUSIONS: I. terricola could successfully adjust its membrane structure and function below 60 g l-1 of citric acid. However, for citric acid concentrations above 80 g l-1, its structure and function were dramatically changed, which might result in reduced functionality.

Inhibition of multiple staphylococcal growth states by a small molecule that disrupts membrane fluidity and voltage.

New molecular approaches to disrupting bacterial infections are needed. The bacterial cell membrane is an essential structure with diverse potential lipid and protein targets for antimicrobials. While rapid lysis of the bacterial cell membrane kills bacteria, lytic compounds are generally toxic to whole animals. In contrast, compounds that subtly damage the bacterial cell membrane could disable a microbe, facilitating pathogen clearance by the immune system with limited compound toxicity. A previously described small molecule, D66, terminates Salmonella enterica serotype Typhimurium (S. Typhimurium) infection of macrophages and reduces tissue colonization in mice. The compound dissipates bacterial inner membrane voltage without rapid cell lysis under broth conditions that permeabilize the outer membrane or disable efflux pumps. In standard media, the cell envelope protects Gram-negative bacteria from D66. We evaluated the activity of D66 in Gram-positive bacteria because their distinct envelope structure, specifically the absence of an outer membrane, could facilitate mechanism of action studies. We observed that D66 inhibited Gram-positive bacterial cell growth, rapidly increased Staphylococcus aureus membrane fluidity, and disrupted membrane voltage while barrier function remained intact. The compound also prevented planktonic staphylococcus from forming biofilms and a disturbed three-dimensional structure in 1-day-old biofilms. D66 furthermore reduced the survival of staphylococcal persister cells and of intracellular S. aureus. These data indicate that staphylococcal cells in multiple growth states germane to infection are susceptible to changes in lipid packing and membrane conductivity. Thus, agents that subtly damage bacterial cell membranes could have utility in preventing or treating disease.IMPORTANCEAn underutilized potential antibacterial target is the cell membrane, which supports or associates with approximately half of bacterial proteins and has a phospholipid makeup distinct from mammalian cell membranes. Previously, an experimental small molecule, D66, was shown to subtly damage Gram-negative bacterial cell membranes and to disrupt infection of mammalian cells. Here, we show that D66 increases the fluidity of Gram-positive bacterial cell membranes, dissipates membrane voltage, and inhibits the human pathogen Staphylococcus aureus in several infection-relevant growth states. Thus, compounds that cause membrane damage without lysing cells could be useful for mitigating infections caused by S. aureus.

Comparative differences in maintaining membrane fluidity and remodeling cell wall between Glycine soja and Glycine max leaves under drought.

Water shortage is one of the most important environmental factors limiting crop yield. In this study, we used wild soybean (Glycine soja Sieb. et Zucc.) and soybean (Glycinemax (L.) Merr.) seedlings as experimental materials, simulated drought stress using soil gravimetry, measured growth and physiological parameters, and analyzed differentially expressed genes and metabolites in the leaves of seedling by integrated transcriptomics and metabolomics techniques. The results indicate that under water deficit, Glycine soja maintained stable photosynthate by accumulating Mg2+, Fe3+, Mn2+, Zn2+ and B3+, and improved water absorption by increasing root growth. Notably, Glycine soja enhanced linoleic acid metabolism and plasma membrane intrinsic protein (PIP1) gene expression to maintain membrane fluidity, and increased pentose, glucuronate and galactose metabolism and thaumatin protein genes expression to remodel the cell wall, thereby increasing water-absorption to better tolerate to drought stress. In addition, it was found that secondary phenolic metabolism, such as phenylpropane biosynthesis, flavonoid biosynthesis and ascobate and aldarate metabolism were weakened, resulting in the collapse of the antioxidant system, which was the main reason for the sensitivity of Glycine max to drought stress. These results provide new insights into plant adaptation to water deficit and offer a theoretical basis for breeding soybean varieties with drought tolerance.

A Sigma1 Receptor Agonist Alters Fluidity and Stability of Lipid Monolayers.

Interactions between the sigma1 receptor agonist PRE-084 and various lipid monolayers, including dipalmitoylphosphatidylcholine (DPPC), DPP-ethanolamine (DPPE), DPP-glycerol (DPPG), DPP-serine (DPPS), palmitoylsphingomyelin (PSM), and cholesterol (Ch), were investigated to elucidate the effects of PRE-084 on membrane fluidity and stability. Their interactions with sigma1 receptor agonists have potential implications for neuroprotection, antidepressant, analgesic, and cognitive enhancement effects. In this study, we observed that the presence of PRE-084 in the subphase led to increased fluidity in DPPC and DPPE monolayers, whereas decreasing fluidity was observed in DPPG, DPPS, and PSM monolayers. The interaction of PRE-084 with Ch monolayers was found to be distinct from its interaction with other lipids. Fluorescence microscopy images revealed changes in the size and shape of liquid-condensed domains in the presence of PRE-084, supporting the notion of altered membrane fluidity. Our findings provide new insights into the interaction of PRE-084 with lipid monolayers and its potential implications for biological and membrane science.

Regulation of meiotic telomere dynamics through membrane fluidity promoted by AdipoR2-ELOVL2.

The cellular membrane in male meiotic germ cells contains a unique class of phospholipids and sphingolipids that is required for male reproduction. Here, we show that a conserved membrane fluidity sensor, AdipoR2, regulates the meiosis-specific lipidome in mouse testes by promoting the synthesis of sphingolipids containing very-long-chain polyunsaturated fatty acids (VLC-PUFAs). AdipoR2 upregulates the expression of a fatty acid elongase, ELOVL2, both transcriptionally and post-transcriptionally, to synthesize VLC-PUFA. The depletion of VLC-PUFAs and subsequent accumulation of palmitic acid in AdipoR2 knockout testes stiffens the cellular membrane and causes the invagination of the nuclear envelope. This condition impairs the nuclear peripheral distribution of meiotic telomeres, leading to errors in homologous synapsis and recombination. Further, the stiffened membrane impairs the formation of intercellular bridges and the germ cell syncytium, which disrupts the orderly arrangement of cell types within the seminiferous tubules. According to our findings we propose a framework in which the highly-fluid membrane microenvironment shaped by AdipoR2-ELOVL2 underpins meiosis-specific chromosome dynamics in testes.

Effect of Lipid Composition on the Interaction of Liposomes with THP-1-Derived Macrophages.

Recently, liposomal formulations that target macrophages have been used to treat lung diseases. However, the detailed mechanism of the cellular uptake must be elucidated to identify a formulation with excellent cellular uptake efficiency to treat non-tuberculous mycobacterial lung disease. We studied the effect of lipid composition on the cellular uptake of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/cholesterol (Chol) liposomes with a size of approximately 200 nm into THP-1-derived macrophages. The amount of DPPC/Chol liposomes (80/20 mol%) was greater than that of DPPC/Chol (60/40 mol%) and DPPC/Chol (67/33 mol%) liposomes. The anisotropy of 1,6-diphenyl-1,3,5-hexatriene indicated that the membrane fluidity of the DPPC/Chol (80/20 mol%) liposomes was higher than that of the other two liposomes. DPPC/Chol (80/20 mol%) and DPPC/Chol (67/33 mol%) liposomes were taken up via clathrin- and caveolae-mediated endocytosis and phagocytosis. However, proteins involved in cellular uptake through ligand-receptor interactions were adsorbed to a greater extent on DPPC/Chol (80/20 mol%) liposomes than on DPPC/Chol (67/33 mol%) liposomes. Pretreatment of cells with antibodies against the low-density lipoprotein receptor and scavenger receptor type B1 largely inhibited the uptake efficiency of DPPC/Chol (80/20 mol%) liposomes. Our results indicate that the membrane fluidity of DPPC/Chol liposomes, which is controlled by the Chol ratio, is an important factor in controlling protein adsorption and the subsequent uptake efficiency of liposomes.

Quantifying Fluorescence Lifetime Responsiveness of Environment-Sensitive Probes for Membrane Fluidity Measurements.

The structural diversity of different lipid species within the membrane defines its biophysical properties such as membrane fluidity, phase transition, curvature, charge distribution, and tension. Environment-sensitive probes, which change their spectral properties in response to their surrounding milieu, have greatly contributed to our understanding of such biophysical properties. To realize the full potential of these probes and avoid misinterpretation of their spectral responses, a detailed investigation of their fluorescence characteristics in different environments is necessary. Here, we examined the fluorescence lifetime of two newly developed membrane order probes, NR12S and NR12A, in response to alterations in their environments such as the degree of lipid saturation, cholesterol content, double bond position and configuration, and phospholipid headgroup. As a comparison, we investigated the lifetime sensitivity of the membrane tension probe Flipper in these environments. Applying fluorescence lifetime imaging microscopy (FLIM) in both model membranes and biological membranes, all probes distinguished membrane phases by lifetime but exhibited different lifetime sensitivities to varying membrane biophysical properties (e.g., cholesterol). While the lifetime of Flipper is particularly sensitive to the membrane cholesterol content, the NR12S and NR12A lifetimes are moderately sensitive to both the cholesterol content and lipid acyl chains. Moreover, all of the probes exhibit longer lifetimes at longer emission wavelengths in membranes of any complexity. This emission wavelength dependency results in varying lifetime resolutions at different spectral regions, which are highly relevant for FLIM data acquisition. Our data provide valuable insights on how to perform FLIM with these probes and highlight both their potential and limitations.

Membrane fluidity homeostasis is required for tobramycin-enhanced biofilm in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen, which causes chronic infections, especially in cystic fibrosis (CF) patients where it colonizes the lungs via the build-up of biofilms. Tobramycin, an aminoglycoside, is often used to treat P. aeruginosa infections in CF patients. Tobramycin at sub-minimal inhibitory concentrations enhances both biofilm biomass and thickness in vitro; however, the mechanism(s) involved are still unknown. Herein, we show that tobramycin increases the expression and activity of SigX, an extracytoplasmic sigma factor known to be involved in the biosynthesis of membrane lipids and membrane fluidity homeostasis. The biofilm enhancement by tobramycin is not observed in a sigX mutant, and the sigX mutant displays increased membrane stiffness. Remarkably, the addition of polysorbate 80 increases membrane fluidity of sigX-mutant cells in biofilm, restoring the tobramycin-enhanced biofilm formation. Our results suggest the involvement of membrane fluidity homeostasis in biofilm development upon tobramycin exposure.IMPORTANCEPrevious studies have shown that sub-lethal concentrations of tobramycin led to an increase biofilm formation in the case of infections with the opportunistic pathogen Pseudomonas aeruginosa. We show that the mechanism involved in this phenotype relies on the cell envelope stress response, triggered by the extracytoplasmic sigma factor SigX. This phenotype was abolished in a sigX-mutant strain. Remarkably, we show that increasing the membrane fluidity of the mutant strain is sufficient to restore the effect of tobramycin. Altogether, our data suggest the involvement of membrane fluidity homeostasis in biofilm development upon tobramycin exposure.

Effects of grafted polymers on the lipid membrane fluidity.

Since the membrane fluidity controls the cellular functions, it is important to identify the factors that determine the cell membrane viscosity. Cell membranes are composed of not only lipids and proteins but also polysaccharide chain-anchored molecules, such as glycolipids. To reveal the effects of grafted polymers on the membrane fluidity, in this study, we measured the membrane viscosity of polymer-grafted giant unilamellar vesicles (GUVs), which were prepared by introducing the poly (ethylene glycol) (PEG)-anchored lipids to the ternary GUVs composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol. The membrane viscosity was obtained from the velocity field on the GUV generated by applying a point force, based on the hydrodynamic model proposed by Henle and Levine. The velocity field was visualized by a motion of the circular liquid ordered (Lo) domains formed by a phase separation. With increasing PEG density, the membrane viscosity of PEG-grafted GUVs increased gradually in the mushroom region and significantly in the brush region. We propose a hydrodynamic model that includes the excluded volume effect of PEG chains to explain the increase in membrane viscosity in the mushroom region. This work provides a basic understanding of how grafted polymers affect the membrane fluidity.