Phospholipid Monolayers Research Articles

Determination of the Coverage of Phosphatidylcholine Monolayers Formed at Silicone Oil–Water Interfaces by Vesicle Fusion

Phospholipid monolayers at oil-water interfaces are used for various biological applications and often formed by vesicle adsorption. However, the adsorbed structures are not well characterized; therefore, fundamental investigation on vesicle adsorption behavior is necessary for correct understanding of the monolayer systems. Herein, we investigated the adsorption of phosphatidylcholine vesicles onto silicone oil-water interfaces using fluorescence microscopy and pendant drop tensiometry. The interfacial monolayer coverage, S, was determined by assuming S = 1 for tightly packed monolayers. Adsorption for 10 min with a lipid concentration of 0.2 mM resulted in S ≈ 0.4. An increase in lipid concentration (0.5-2 mM) and adsorption time (1 h) moderately increased monolayer coverage (S ≈ 0.6). However, extended adsorption for 24 h only slightly increased coverage (S ≈ 0.7). Monolayers with an S < 0.6 were homogeneous, while those with an S > 0.6 were associated with several vesicular structures. The surface density of these bound vesicles increased with increasing S. We conclude that vesicles readily fused with the interface to form monolayers at S < 0.6 and that their fusogenicity considerably decreased at S > 0.6. These results demonstrate that the ability of vesicles to form monolayers is determined by the interfacial coverage.

Abstract 2562: Causes and consequences of adiposomogenesis in breast cancer cells

Abstract Malignant tumors exhibit altered metabolism resulting in a highly acidic extracellular microenvironment. Adaptation to acidic conditions is a pre-requisite for tumor cells to survive and thrive and to out-compete the stroma into which they invade. Acid adaptation has been associated with chronic activation of autophagy and redistribution of the lysosomal proteins to the plasma membrane. In addition to these survival mechanisms, tumor cells under acidic conditions accumulate cytoplasmic lipid droplets (adiposomes); dynamic organelles that store neutral lipids surrounded by a shell of perilipin (PLIN) proteins and a phospholipid monolayer. Adiposomes are dynamic organelles that store neutral lipids surrounded by a shell of proteins (PLIN2) and a phospholipid monolayer. High expression of PLIN2 was observed to be strongly associated with poor overall survival in breast cancer patients. In vitro, breast cancer cells rapidly and robustly accumulated adiposomes when grown in acidic media revealed by nile red and PLIN-2 staining. The acid-induced lipogenic phenotype persists even when the cells are grown in de-lipidated serum, indicating that the source of lipids is de-novo and endogenous. Adiposome formation at low pH was attenuated when cells were treated with inhibitors of fatty acid synthesis, FAS; such as TOFA or C75. Further, these inhibitors were selectively cytotoxic under acidic conditions indicating that adiposomogenesis is a survival mechanism. Consistent with increased FAS we also observed using 13C isotopomer analysis a major shift in glucose metabolism from Embden Meyerhof to the Pentose Phosphate Pathway, which results in increased production of NADPH, necessary for de novo lipid synthesis. In addition, we observed that cells at low pH had higher rates of oxygen consumption (OCR) compared to controls using Seahorse profiling. The major source of the lipid precursors was identified to be autophagy-derived ketogenic amino acids using LC-LS/MS following 13C tracer pre-incubation. Further, we tested the hypothesis that adiposomogenesis is induced by acid signal and this signaling is mediated by one (or more) acid sensing G-protein coupled receptors. Both OGR1 and TDAG8 were strongly expressed in our systems. CRISPR/Cas9 mediated depletion of these receptors showed that TDAG8 KO had no effect, but that KO of OGR1 abrogated adiposome accumulation under acidic conditions in MCF7 and T47D cells. Further, OGR1 knockout cells were defective in acid induced autophagy. In xenograft models, OGR1 knockout MCF7 cells formed significantly smaller tumors compared to control cells. Hence, accumulation of adiposomes is a highly regulated process related to storing autophagic products, and appears to be important in cell survival in acid stress. This increased dependence on lipid metabolism revealed novel therapeutic vulnerabilities Citation Format: Smitha R. Pillai, Iqbal Mahmud, Michael Langsen, Jonathan Wojtkowiak, Jonathan Nguyen, Marylin Bui, Robert Gatenby, Timothy Garrett, Robert Gillies. Causes and consequences of adiposomogenesis in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2562.

Abstract 5130: Proteomic characterization of cytoplasmic lipid droplets in human metastatic breast cancer cells

Abstract One of the characteristic features of metastatic breast cancer is altered lipid metabolism, including increased cellular storage of neutral lipid in cytoplasmic lipid droplets (CLDs). CLD accumulation is associated with increased cancer aggressiveness, suggesting CLDs contribute to metastasis. Although multiple hypotheses exist for how CLDs may promote cancer progression, how exactly CLDs contribute to breast cancer metastasis is not clear. CLDs are composed of a neutral lipid core, a phospholipid monolayer, and associated proteins. Proteins that associate with CLDs are thought to regulate CLD metabolism; however, the proteome of CLDs in metastatic breast cancer and how these proteins may contribute to altered lipid metabolism and breast cancer progression is unknown. Therefore, we utilized untargeted shotgun proteomics and transmission electron microscopy to identify the proteome and assess the characteristics of CLDs in the MCF10CA1a human metastatic breast cancer cell line. We identified a total number of 1537 proteins in the CLD fraction isolated from MCF10CA1a cells, and these proteins were associated with a variety of cellular processes. Interestingly, unlike other cell lines such as adipocytes or enterocytes, the most enriched protein categories were involved in cellular processes outside of lipid metabolism. For example, proteins involved in cell-cell adhesion were the most enriched in the CLD fraction, and some of these proteins had associated terms in cell migration and signaling. In addition, we characterized CLD size and area in MCF10CA1a cells using transmission electron microscopy. Our results provide a hypothesis-generating list of potential players in breast cancer progression and aggressiveness, making them possible therapeutic targets for prevention or treatment of this disease. Citation Format: Alyssa S. Zembroski, Chaylen Andolino, Kimberly K. Buhman, Dorothy Teegarden. Proteomic characterization of cytoplasmic lipid droplets in human metastatic breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5130.

Triacylglycerols sequester monotopic membrane proteins to lipid droplets

Triacylglycerols (TG) are synthesized at the endoplasmic reticulum (ER) bilayer and packaged into organelles called lipid droplets (LDs). LDs are covered by a single phospholipid monolayer contiguous with the ER bilayer. This connection is used by several monotopic integral membrane proteins, with hydrophobic membrane association domains (HDs), to diffuse between the organelles. However, how proteins partition between ER and LDs is not understood. Here, we employed synthetic model systems and found that HD-containing proteins strongly prefer monolayers and returning to the bilayer is unfavorable. This preference for monolayers is due to a higher affinity of HDs for TG over membrane phospholipids. Protein distribution is regulated by PC/PE ratio via alterations in monolayer packing and HD-TG interaction. Thus, HD-containing proteins appear to non-specifically accumulate to the LD surface. In cells, protein editing mechanisms at the ER membrane would be necessary to prevent unspecific relocation of HD-containing proteins to LDs.

Novel Drug Formulation for the Treatment of Hepatic Cancer- A Review

For metabolic transformation, uptake, detoxification, and excretion liver is the primary organ that is highly equipped. Thus, the liver requires targeting by means like a carrier-mediated mechanism to take xenobiotics into the bile, though high hepatic concentration is achieved by most of the drugs. Thus resulting in high first-pass metabolism displayed by the drugs and thus resulting in rapid clearance of the drugs. Uptake of particulate materials is highly contributed by the kupffer cells largely. However, drug uptake by the liver is highly dependent on hepatocytes. In drug delivery, tissue engineering and regenerative medicines which are various biomedical applications construction of nanoscale based bioactive materials is a desirable approach of self-assembly. By using a targeting moiety, we can decrease the side effects of the drug and increases the therapeutic effect of the drug. Lipoproteins are potential drug carrier to target the organs. Lipoproteins consist of cholesterol, polar lipid core surrounding phospholipid monolayer and apoproteins are embedded in it, and these lipoproteins are spherical. The core is a polar lipid in nature so that highly hydrophobic drugs are easily incorporated into the core. Lipoproteins are completely non-immunogenic, biodegradable nature. The present review should be regularly inspected to beat into the global market at an affordable price as well, particularly the vehicles which are proven to be efficacious in drug delivery systems used to treat liver diseases like cancer.

Chain Cleavage of Bioinspired Bacterial Membranes Photoinduced by Eosin Decyl Ester.

Photodynamic therapy (PDT) is promising for bacterial inactivation since cellular internalization of photosensitizers (PS) is not crucial for the treatment effectiveness. Photoinduced damage in the lipid envelope may already induce microbial inactivation, which requires PS capable of easily penetrating into the membrane. Herein, we report on the insertion of the PS eosin decyl ester (EosDec) into Langmuir films of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), and cardiolipin (CLP) used as mimetic systems of bacterial membranes. Surface pressure isotherms and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) indicated that the hydrophobic nature of EosDec favored deeper penetration in all the phospholipid monolayers. The incorporation of EosDec led to monolayer expansion, especially in the anionic DOPG and CLP owing to repulsive electrostatic interactions, and induced disorder in the lipid chains. Light irradiation of DOPE, DOPG, and CLP monolayers containing EosDec increased the rate of material loss to the subphase, which is attributed to cleavage of lipid chains triggered by contact-dependent reactions between excited states of EosDec and lipid unsaturations. The latter is key for membrane permeabilization and efficiency in microbial inactivation.

A general approach to maximise information density in neutron reflectometry analysis

Neutron and x-ray reflectometry are powerful techniques facilitating the study of the structure of interfacial materials. The analysis of these techniques is ill-posed in nature requiring the application of model-dependent methods. This can lead to the over- and under- analysis of experimental data when too many or too few parameters are allowed to vary in the model. In this work, we outline a robust and generic framework for the determination of the set of free parameters that are capable of maximising the information density of the model. This framework involves the determination of the Bayesian evidence for each permutation of free parameters; and is applied to a simple phospholipid monolayer. We believe this framework should become an important component in reflectometry data analysis and hope others more regularly consider the relative evidence for their analytical models.

Implications of lipid droplets in lung cancer: Associations with drug resistance.

Cancer cells usually show different metabolic patterns compared with healthy cells due to the reprogramming of metabolic processes. The process of lipid metabolism undergoes notable changes, leading to the accumulation of lipid droplets in cells. Additionally, this phenotype is considered an important marker of cancer cells. Lipid droplets are a highly dynamic type of organelle in the cell, which is composed of a neutral lipid core, a monolayer phospholipid membrane and lipid droplet-related proteins. Lipid droplets are involved in several biological processes, including cell proliferation, apoptosis, lipid metabolism, stress, immunity, signal transduction and protein trafficking. Epidermal growth factor receptor (EGFR)-activating mutations are currently the most effective therapeutic targets for non-small cell lung cancer. Several EGFR tyrosine kinase inhibitors (EGFR-TKIs) that target these mutations, including gefitinib, erlotinib, afatinib and osimertinib, have been widely used clinically. However, the development of acquired resistance has a major impact on the efficacy of these drugs. A number of previous studies have reported that the expression of lipid droplets in the tumor tissues of patients with lung cancer are elevated, whereas the association between elevated numbers of lipid droplets and drug resistance has received little attention. The present review describes the potential association between lipid droplets and drug resistance. Furthermore, the mechanisms and implications of lipid droplet accumulation in cancer cells are analyzed, as wells as the mechanism by which lipid droplets suppress endoplasmic reticulum stress and apoptosis, which are essential for the development and treatment of lung cancer.

196 Relationship between the physicochemical effect of compounds on phospholipid membranes and their influence on epidermal permeability barrier homeostasis

Secretion of lipids from lamellar bodies into the intercellular space between stratum granulosum and stratum corneum is a crucial step in epidermal water-impermeable barrier homeostasis. Our previous work showed that topical application of fatty acids, sex hormones, hexoses, polyols and polymers influences barrier homeostasis, but the effects are highly dependent on even small variations of molecular structure. Therefore, we evaluated the effects of these molecules on the physicochemical properties of phospholipid monolayers and liposomes as models of the lamellar body membrane and cell membrane.

Characteristic responses of a 1,2-dioleoyl-sn-glycero-3-phosphocholine molecular layer to monovalent and divalent metal cations

Monovalent and divalent cations were added to a phospholipid (1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)) solution in order to evaluate the effect of metal cations on the phospholipid monolayer. While the surface pressure-area (Π-A) isotherm of the phospholipid monolayer at the air-water interface did not change when monovalent cations (Na+, K+) were added, Π increased with the addition of divalent cations (Mg2+, Ca2+, Sr2+) according to their atomic numbers. Fourier-transform infrared spectroscopy and differential scanning calorimetry were used to evaluate the interactions between DOPC and cations. Experimental results suggest that cation valence and atomic number play an important role in the characteristic interactions with DOPC.

Impeded Molecular Reorganization by Polyethylene Glycol Conjugation Revealed by X-ray Reflectivity and Diffraction Measurements.

Polyethylene glycol (PEG) coatings have been widely applied in pharmaceutical and biomedical systems to prevent nonspecific protein absorption, increase vesicle blood circulation time, and sustain drug release. This study systematically investigated the planar interfacial organization of phospholipid monolayers containing various amounts of PEG conjugations before and after enzyme-catalyzed degradation of the lipids using X-ray reflectivity and grazing incidence X-ray diffraction techniques. Results showed that attaching PEG to the headgroup of the lipids up to 15 mol % had limited effects on molecular packing of the lipid monolayers in the condensed phase at the gas-liquid interface and negligible effects on the enzyme adsorption to the interface. After enzyme-catalyzed degradation, equimolar fatty acids and lyso PC were generated. The fatty acids together with the subphase Ca2+ self-assembled into highly organized multilayer domains at the interface. The X-ray measurements unambiguously revealed that the densely packed PEG markedly hindered microphase separation and formation of the palmitic acid-Ca2+ complexes.

In Situ Monitoring of the Unsaturated Phospholipid Monolayer Oxidation in Ambient Air by HD-SFG Spectroscopy

The pulmonary surfactant monolayer is indispensable for the respiratory system. Recently, it was reported that some unsaturated lipids of the pulmonary surfactants are oxidized by low-level ozone in ambient air. However, the molecular-level understanding of the reaction mechanism is still limited due to technical difficulties. We applied heterodyne-detected sum frequency generation (HD-SFG) spectroscopy to probe the reaction process of an unsaturated phospholipid monolayer (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, POPC), which is one of the major lipids in the pulmonary surfactant, under low-level ozone (30 ± 5 ppb). The HD-SFG spectroscopy realized the accurate peak assignments of the spectra and the identification of molecular species with high sensitivity, which were impossible with previous measurements. The time-resolved spectra indicated that the C═C moiety in the unsaturated alkyl chain is selectively oxidized by ozone with a time constant of 22 ± 3 min by first-order reaction kinetics. Furthermore, it was revealed for the first time that the reaction product of the POPC monolayer under low-level ozone is not the carboxylic form but the aldehyde form based on the vibrational spectroscopy results. The present study has deepened our molecular-level understanding of the oxidation mechanism of unsaturated lipids that are widely found in many biological systems.

Control of Director Fields in Phospholipid-Coated Liquid Crystal Droplets.

In liquid crystal (LC) droplets, small changes in surface anchoring energy can produce large changes in the director field which result in readily detectable optical effects. This makes them attractive for use as biosensors. Coating LC droplets with a phospholipid monolayer provides a bridge between the hydrophobic world of LCs and the water-based world of biology and makes it possible to incorporate naturally occurring biosensor systems. However, phospholipids promote strong perpendicular (homeotropic) anchoring that can inhibit switching of the director field. We show that the tendency for phospholipid layers to promote perpendicular anchoring can be suppressed by using synthetic phospholipids in which the acyl chains are terminated with bulky tert-butyl or ferrocenyl groups; the larger these end-group(s), the less likely the system is to be perpendicular/radial. Additionally, the droplet director field is found to be dependent on the nature of the LC, particularly its intrinsic surface properties, but not (apparently) on the sign of the dielectric anisotropy, the proximity to the melting/isotropic phase transition, the surface tension (in air), or the values of the Frank elastic constants.

Protein Binding to Artificial Lipid Droplets Demonstrates the Importance of Phospholipid Composition

All cells and tissues store excess lipid in intracellular structures known as lipid droplets. Each lipid droplet consists of a neutral lipid core (primarily made of triacylglyceride and cholesteryl ester) surrounded by a phospholipid monolayer. Several proteins interact specifically with the surface of the lipid droplet and control their growth and degradation. Here, we share the development of an in vitro binding assay, that utilizes synthetic lipid droplets and partially purified lipid droplet proteins, to study protein binding to lipid droplets. We separate bound from unbound proteins with sucrose gradient ultracentrifugation and detect lipid droplet proteins following SDS‐PAGE and western blotting. We have used this assay to show that changing the phospholipid composition of the lipid droplet surface alters interactions with perilipin 2, a ubiquitously expressed lipid droplet protein that is predicted to interact with lipid droplets through an amphipathic alpha helix. Specifically, perilipin 2 association with synthetic lipid droplets was increased when the amount of phosphatidylethanolamine relative to phosphatidylcholine was increased. We hypothesize that the smaller headgroups of phosphatidylethanolamine allows additional interaction with the underlying neutral lipid. These results may be relevant to the formation of lipid droplets in alcoholic fatty liver disease, where lipid droplets form with altered levels of phosphatidylethanolamine and phosphatidylcholine.Support or Funding InformationFunding for this research was provided by an ASBMB Undergraduate Research Award and the Lowell and Doris Kispert Endowment at St. Olaf College.

Probing the Nature of the Hydrophobic Cleft of Perilipin 5

Numerous conditions that currently threaten human health are related to aberrant lipid storage. These conditions include type II diabetes mellitus, non‐alcoholic fatty liver disease, obesity and heart disease. Nearly all cells of the body are able to store neutral lipids but this storage is not without consequence. Neutral lipids are stored in the cell in specific organelles termed lipid storage droplets. These organelles consist of a hydrophobic core surrounded by a phospholipid monolayer studded with several proteins that regulate the formation and breakdown of the droplet. Perilipin 5 is a lipid droplet protein which has been demonstrated to play key roles in lipid storage and mobilization in oxidative tissues including cardiac muscle and liver tissue in the fasted state. Perilipin 5 is a member of the perilipin protein family, a family encoded by five highly related genes. A different family member, perilipin 3, has had its structure solved. The C‐terminus of perilipin 3 consists of a four‐helix bundle domain and a mixed alpha‐beta structure. At the interface of these two units is a hydrophobic cleft. We have aligned the carboxy terminal region of perilipin 5 with perilipin 3 and have used threading and molecular modeling to generate a theoretical model of the region in perilipin 5. This structure retains the four‐helix bundle, mixed alpha‐beta domain and the hydrophobic cleft of perilipin 3, albeit with a potentially different amino acid sequence. The perilipin 5 structure differs in that the cleft contains a short insertion which may affect this pocket. Ongoing studies seek to generate mutations to this cleft to probe the function of this structural feature in perilipin 5.

Protein Binding to Lipid Droplets with Altered Ratios of Surface Phospholipids

Alcoholic Fatty Liver Disease (AFLD) is characterized by an increase in lipid droplets in the liver after acute ingestion of alcohol. Previous experiments with a rat model of AFLD show hepatic lipid droplets contain altered levels of the predominant classes of phospholipids. Specifically, hepatic lipid droplets from AFLD rats were found with lower amounts of phosphatidylcholine (PC) relative to phosphatidylethanolamine (PE). Our experiments aim to determine if the altered phospholipid composition of AFLD lipid droplets causes differences in the numbers or types of lipid droplet‐associated proteins. We selected proteins based on their membrane binding motifs and roles in lipid droplet homeostasis. AML12 cells cultured in choline‐deficient media simulate the altered phospholipid composition of AFLD lipid droplets. We isolated lipid droplets from AML12 cells cultured with and without choline and examined proteins of interest by SDS‐PAGE and western blotting. We found equivalent levels of rab18 and lanosterol synthase in lipid droplet fractions from cells grown with and without choline. Alternatively, perilipin 1, perilipin 2, and CIDE‐C are predominantly found in lipid droplet fractions from cells grown without choline (with lower ratios of PC:PE). The proteins that are most influenced by lipid droplet surface composition are all predicted to associate with lipid droplets through amphipathic alpha helices. By evaluating how altered phospholipid monolayer composition affects the binding of lipid droplet proteins, we seek to further elucidate the biochemical pathways that contribute to AFLD.Support or Funding InformationThis research was supported by St. Olaf College and the TRIO McNair Scholars Program.

Confocal Raman Microscopy Investigation of Phospholipid Monolayers Deposited on Nitrile-Modified Surfaces in Porous Silica Particles.

Phospholipid bilayers deposited on a variety of surfaces provide models for investigation of the lipid membrane structure and supports for biocompatible sensors. Hybrid-supported phospholipid bilayers (HSLBs) are stable membrane models for these investigations, typically prepared by self-assembly of a lipid monolayer over an n-alkane-modified surface. HSLBs have been prepared on n-alkyl chain-modified silica and used for lipophilicity-based chromatographic separations. The structure of these hybrid bilayers differs from vesicle membranes where the lipid head group spacing is greater due to interdigitation of the lipid acyl chains with the underlying n-alkyl chains bound to the silica surface. This interdigitated structure exhibits a broader melting transition at a higher temperature due to strong interactions between the lipid acyl chains and the immobile n-alkyl chains bound to silica. In the present work, we seek to reduce the interactions between a lipid monolayer and its supporting substrate by self-assembly of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) on porous silica functionalized with nitrile-terminated surface ligands. The frequency of Raman scattering of the surface -C≡N stretching mode at the lipid-nitrile interface is consistent with an n-alkane-like environment and insensitive to lipid head group charge, indicating that the lipid acyl chains are in contact with the surface nitrile groups. The head group area of this lipid monolayer was determined from the within-particle phospholipid concentration and silica specific surface area and found to be 54 ± 2 Å2, equivalent to the head group area of a DMPC vesicle bilayer. The structure of these nitrile-supported phospholipid monolayers was characterized below and above their melting transition by confocal Raman microscopy and found to be nearly identical to DMPC vesicle bilayers. Their narrow gel-to-fluid-phase melting transition is equivalent to dispersed DMPC vesicles, suggesting that the acyl chain structure on the nitrile support mimics the outer leaflet structure of a vesicle membrane.

Neutral lipids regulate amphipathic helix affinity for model lipid droplets

Cellular lipid droplets (LDs) have a neutral lipid core shielded from the aqueous environment by a phospholipid monolayer containing proteins. These proteins define the biological functions of LDs, and most of them bear amphipathic helices (AH), which can selectively target to LDs, or to LD subsets. How such binding preference happens remains poorly understood. Here, we found that artificial LDs made of different neutral lipids but presenting equal phospholipid packing densities differentially recruit AHs. Varying the phospholipid density shifts the binding levels, but the differential recruitment is unchanged. We found that the binding level of AHs is defined by their interaction preference with neutral lipids and ability to decrease surface tension. The phospholipid packing level regulates mainly the amount of neutral lipid accessible. Therefore, it is the hydrophobic nature of the phospholipid packing voids that controls the binding level of AHs. Our data bring us a major step closer to understanding the binding selectivity of AHs to lipid membranes.

Can simple physicochemical studies predict the effects of molecules on epidermal water‐impermeable barrier function?

Improvement of the water-impermeable barrier function of skin is clinically important, because barrier abnormality is associated with various skin diseases, such as psoriasis or atopic dermatitis. We have shown that topical application of fatty acids, sex hormones, hexoses, polyols and polymers influences barrier homeostasis, but the effects are highly dependent on even small variations of molecular structure. Moreover, the effects appear within one hour after application and thus are likely to be non-genomic (physicochemical) phenomena. Secretion of lipids from lamellar bodies into the intercellular space between stratum granulosum and stratum corneum is a crucial step in epidermal water-impermeable barrier homeostasis, especially at the early stage of barrier recovery after damage, and phase transition of the lipid lamellar structure in the epidermis is an important part of this process. Therefore, we evaluated the effects of the above molecules on the physicochemical properties of phospholipid monolayers and liposomes as models of the lamellar body membrane and cell membrane. Molecules that influenced the barrier recovery process also altered the stability of liposomes and the air-water surface pressure of phospholipid monolayers. Studies using attenuated total reflection Fourier-transform infrared spectroscopy (ATR FT-IR), differential scanning calorimetry (DSC) and 13 C nuclear magnetic resonance (NMR) spectrometry suggested that molecules influencing barrier recovery interact specifically with phospholipids. The idea that molecules interacting with phospholipids may influence barrier homeostasis should open up new approaches to the treatment of a variety of skin diseases.

Live-Cell Superresolution Microscopy of Faa4 Re-Distribution on Lipid Droplets during Metabolic Transitions in Yeast

Lipid droplets (LDs) are energy storage organelles of a cell with a neutral lipid core surrounded by a phospholipid monolayer. Cells achieve their lipid and energy homeostasis by actively regulating the synthesis and the breakdown of neutral lipids stored in LDs during metabolic transitions. In order to accommodate the change in size, number and neutral lipid content of LDs, cells regulate the abundance and the density of specific proteins involved in the synthesis and the breakdown of neutral lipids. In yeast, Faa4 encodes for one of the fatty acyl-CoA synthetases required for the activation of fatty acids essential for making neutral lipids. In this study, we employ correlative Photo-Activated Localization Microscopy (PALM) and conventional fluorescence microscopy to resolve and quantify the density of Faa4 on LDs below the optical diffraction limit, on the ER, and on the vacuole as yeast cells transition between exponential, stationary and lag phases based on the nutrient availability. We find that the Faa4 density on LDs increases as LDs grow in size and re-localize to the vacuolar membrane from ER when the cell progresses to the stationary phase from the exponential growth phase. However, Faa4 is predominately localized to vacuolar lumen within ∼2 hrs of dilution in fresh media suggesting rapid consumption of LDs upon growth resumption. The increase in Faa4 density on LDs that are about to enter the vacuole for breakdown may therefore be the major mechanism how Faa4 shuttles into the vacuole to activate fatty acids. Our approach to resolve and quantify the density of regulatory proteins on LDs in living cells may help in future studies to better understand how cells differentially regulate their density to maintain lipid and energy homeostasis based on the metabolic needs.