Membrane Lipid Organization Research Articles

Septin assemblies promote the lipid organization of membranes.

Cytoskeletal-mediated membrane compartmentalization is essential to support cellular functions, from signaling to cell division, migration, or phagocytosis. Septins are cytoskeletal proteins that directly interact with membranes, acting as scaffolds to recruit proteins to cellular locations and as structural diffusion barriers. How septins interact with and remodel the lipid organization of membranes is unclear. Here, we combined minimal reconstituted systems and yeast cell imaging to study septin-mediated membrane organization. Our results show that at low concentrations membrane-diffusive septins self-assemble into sub-micrometric patches that co-exist with the septin collar at the division site. We found that patches are made of short septin filaments and that are able to modulate the lipid organization of membranes. Furthermore, we show that the polybasic domain of Cdc11 influences the membrane-organizing and curvature-sensing properties of septins. Collectively, our work provides understanding of the molecular mechanisms by which septins can support cellular functions intimately linked to membranes.

Halogenated Cholesterol Alters the Phase Behavior of Ternary Lipid Membranes.

Eukaryotic plasma membranes exhibit nanoscale lateral lipid heterogeneity, a feature that is thought to be central to their function. Studying these heterogeneities is challenging since few biophysical methods are capable of detecting domains at submicron length scales. We recently showed that cryogenic electron microscopy (cryo-EM) can directly image nanoscale liquid-liquid phase separation in extruded liposomes due to its ability to resolve the intrinsic thickness and electron density differences of ordered and disordered phases. However, the intensity contrast between these phases is poor compared with conventional fluorescence microscopy and is thus both a limiting factor and a focal point for optimization. Because the fundamental source of intensity contrast is the spatial variation in electron density within the bilayer, lipid modifications aimed at selectively increasing the electron density of one phase might enhance the ability to resolve coexisting phases. To this end, we investigated model membrane mixtures of DPPC/DOPC/cholesterol in which one hydrogen of cholesterol's C19 methyl group was replaced by an electron-rich halogen atom (either bromine or iodine). We characterized the phase behavior as a function of composition and temperature using fluorescence microscopy, Förster resonance energy transfer, and cryo-EM. Our data suggest that halogenated cholesterol variants distribute approximately evenly between liquid-ordered and liquid-disordered phases and are thus ineffective at enhancing the intensity difference between them. Furthermore, replacing more than half of the native cholesterol with halogenated cholesterol variants dramatically reduces the size of the membrane domains. Our results reinforce how small changes in the sterol structure can have a large impact on the lateral organization of membrane lipids.

Coarse-Grained Simulations of Adeno-Associated Virus and Its Receptor Reveal Influences on Membrane Lipid Organization and Curvature.

Adeno-associated virus (AAV) is a well-known gene delivery tool with a wide range of applications, including as a vector for gene therapies. However, the molecular mechanism of its cell entry remains unknown. Here, we performed coarse-grained molecular dynamics simulations of the AAV serotype 2 (AAV2) capsid and the universal AAV receptor (AAVR) in a model plasma membrane environment. Our simulations show that binding of the AAV2 capsid to the membrane induces membrane curvature, along with the recruitment and clustering of GM3 lipids around the AAV2 capsid. We also found that the AAVR binds to the AAV2 capsid at the VR-I loops using its PKD2 and PKD3 domains, whose binding poses differs from previous structural studies. These first molecular-level insights into AAV2 membrane interactions suggest a complex process during the initial phase of AAV2 capsid internalization.

Cellular Output and Physicochemical Properties of the Membrane-Derived Vesicles Depend on Chemical Stimulants.

Synthetic liposomes are widely used as drug delivery vehicles in biomedical treatments, such as for mRNA-based antiviral vaccines like those recently developed against SARS-CoV-2. Extracellular vesicles (EVs), which are naturally produced by cells, have emerged as a next-generation delivery system. However, key questions regarding their origin within cells remain unresolved. In this regard, plasma membrane vesicles (PMVs), which are essentially produced from the cellular plasma membrane (PM), present a promising alternative. Unfortunately, their properties relevant to biomedical applications have not be extensively studied. Therefore, we conducted a thorough investigation of the methods used in the production of PMVs. By leveraging advanced fluorescence techniques in microscopy and flow cytometry, we demonstrated a strong dependence of the physicochemical attributes of PMVs on the chemicals used during their production. Following established protocols employing chemicals such as paraformaldehyde (PFA), N-ethylmaleimide (NEM) or dl-dithiothreitol (DTT) and by developing a modified NEM-based method that involved a hypotonic shock step, we generated PMVs from THP-1 CD1d cells. We systematically compared key parameters such as vesicle output, their size distribution, vesicular content analysis, vesicular membrane lipid organization and the mobility of a transmembrane protein. Our results revealed distinct trends: PMVs isolated using NEM-based protocols closely resembled natural vesicles, whereas PFA induced significant molecular cross-linking, leading to notable changes in the biophysical properties of the vesicles. Furthermore, our novel NEM protocol enhanced the efficiency of PMV production. In conclusion, our study highlights the unique characteristics of chemically produced PMVs and offers insights into their potentially diverse yet valuable biological functions.

Construction and validation of a prognostic model for overall survival time of patients with ovarian cancer by metabolism-related genes.

Ovarian cancer is a female-specific malignancy with high morbidity and mortality. The metabolic reprogramming of tumor cells is closely related to the biological behavior of tumors. The prognostic signature of the metabolism-related gene (MRGs) was established by LASSO-Cox regression analysis. The prognostic signature of MRGs was also prognosticated in each clinical subgroup. These genes were subjected to functional enrichment analysis and tissue expression exploration. Analysis of the MRG prognostic signature in terms of immune cell infiltration and antitumor drug susceptibility was also performed. A MRG prognostic signature including 21 genes was established and validated. Most of the 21 MRGs were expressed at different levels in ovarian cancer than in normal ovarian tissue. The enrichment analysis suggested that MRGs were involved in lipid metabolism, membrane organization, and molecular binding. The MRG prognostic signature demonstrated the predictive value of overall survival time in various clinical subgroups. The monocyte, NKT, Tgd and Tex cell scores showed differences between the groups with high- and low-risk score. The antineoplastic drug analysis we performed provided information on ovarian cancer drug therapy and drug resistance. In vitro experiments verified that PLCH1 in 21 MRGs can regulate the apoptosis and proliferation of ovarian cancer cells. This metabolism-related prognostic signature was a potential prognostic factor in patients with ovarian cancer, demonstrating high stability and accuracy.

Cryo-EM architecture of a near-native stretch-sensitive membrane microdomain

Biological membranes are partitioned into functional zones termed membrane microdomains, which contain specific lipids and proteins1–3. The composition and organization of membrane microdomains remain controversial because few techniques are available that allow the visualization of lipids in situ without disrupting their native behaviour3,4. The yeast eisosome, composed of the BAR-domain proteins Pil1 and Lsp1 (hereafter, Pil1/Lsp1), scaffolds a membrane compartment that senses and responds to mechanical stress by flattening and releasing sequestered factors5–9. Here we isolated near-native eisosomes as helical tubules made up of a lattice of Pil1/Lsp1 bound to plasma membrane lipids, and solved their structures by helical reconstruction. Our structures reveal a striking organization of membrane lipids, and, using in vitro reconstitutions and molecular dynamics simulations, we confirmed the positioning of individual PI(4,5)P2, phosphatidylserine and sterol molecules sequestered beneath the Pil1/Lsp1 coat. Three-dimensional variability analysis of the native-source eisosomes revealed a dynamic stretching of the Pil1/Lsp1 lattice that affects the sequestration of these lipids. Collectively, our results support a mechanism in which stretching of the Pil1/Lsp1 lattice liberates lipids that would otherwise be anchored by the Pil1/Lsp1 coat, and thus provide mechanistic insight into how eisosome BAR-domain proteins create a mechanosensitive membrane microdomain.

Chitosan hybrid nanomaterials: A study on interaction with biomimetic membranes

This study examined the influence of nanomaterials (NMs) on the organization of membrane lipids and the resulting morphological changes. The cell plasma membrane is heterogeneous, featuring specialized lipid domains in the liquid-ordered (Lo) phase surrounded by regions in the liquid-disordered (Ld) phase. We utilized model membranes composed of various lipids and lipid mixtures in different phase states to investigate the interactions between the NMs and membrane lipids. Specifically, we explored the interactions of pure chitosan (CS) and CS-modified nanocomposites (NCs) with ZnO, CuO, and SiO2 with four lipid mixtures: egg-phosphatidylcholine (EggPC), egg-sphingomyelin/cholesterol (EggSM/Chol), EggPC/Chol, and EggPC/EggSM/Chol, which represent the coexistence of Ld, Lo, and Ld/Lo, respectively. The data show that CS NMs increase the membrane lipid order at glycerol level probed by Laurdan spectroscopy. Additionally, the interaction of CS-based NMs with membranes leads to an increase in bending elasticity modulus, zeta potential, and vesicle size. The lipid order changes are most significant in the highly fluid Ld phase, followed by the Lo/Ld coexistence phase, and are less pronounced in the tightly packed Lo phase. CS NMs induced egg PC vesicle adhesion, fusion, and shrinking. In heterogeneous Lo/Ld membranes, inward invaginations and vesicle shrinking via the Ld phase were observed. These findings highlight mechanisms involved in CS NM-lipid interactions in membranes that mimic plasma membrane heterogeneity.

Antimicrobial Peptides Increase Line Tension in Raft-Forming Lipid Membranes.

The formation of phase separated membrane domains is believed to be essential for the function of the cell. The precise composition and physical properties of lipid bilayer domains play crucial roles in regulating protein activity and governing cellular processes. Perturbation of the domain structure in human cells can be related to neurodegenerative diseases and cancer. Lipid rafts are also believed to be essential in bacteria, potentially serving as targets for antibiotics. An important question is how the membrane domain structure is affected by bioactive and therapeutic molecules, such as surface-active peptides, which target cellular membranes. Here we focus on antimicrobial peptides (AMPs), crucial components of the innate immune system, to gain insights into their interaction with model lipid membranes containing domains. Using small-angle neutron/X-ray scattering (SANS/SAXS), we show that the addition of several natural AMPs (indolicidin, LL-37, magainin II, and aurein 2.2) causes substantial growth and restructuring of the domains, which corresponds to increased line tension. Contrast variation SANS and SAXS results demonstrate that the peptide inserts evenly in both phases, and the increased line tension can be related to preferential and concentration dependent thinning of the unsaturated membrane phase. We speculate that the lateral restructuring caused by the AMPs may have important consequences in affecting physiological functions of real cells. This work thus shines important light onto the complex interactions and lateral (re)organization in lipid membranes, which is relevant for a molecular understanding of diseases and the action of antibiotics.

UV-B Radiation Disrupts Membrane Lipid Organization and Suppresses Protein Mobility of GmNARK in Arabidopsis.

While it is well known that plants interpret UV-B as an environmental cue and a potential stressor influencing their growth and development, the specific effects of UV-B-induced oxidative stress on the dynamics of membrane lipids and proteins remain underexplored. Here, we demonstrate that UV-B exposure notably increases the formation of ordered lipid domains on the plasma membrane (PM) and significantly alters the behavior of the Glycine max nodule autoregulation receptor kinase (GmNARK) protein in Arabidopsis leaves. The GmNARK protein was located on the PM and accumulated as small particles in the cytoplasm. We found that UV-B irradiation interrupted the lateral diffusion of GmNARK proteins on the PM. Furthermore, UV-B light decreases the efficiency of surface molecule internalization by clathrin-mediated endocytosis (CME). In brief, UV-B irradiation increased the proportion of the ordered lipid phase and disrupted clathrin-dependent endocytosis; thus, the endocytic trafficking and lateral mobility of GmNARK protein on the plasma membrane are crucial for nodule formation tuning. Our results revealed a novel role of low-intensity UV-B stress in altering the organization of the plasma membrane and the dynamics of membrane-associated proteins.

Hydrophobic mismatch drives self-organization of designer proteins into synthetic membranes

The organization of membrane proteins between and within membrane-bound compartments is critical to cellular function. Yet we lack approaches to regulate this organization in a range of membrane-based materials, such as engineered cells, exosomes, and liposomes. Uncovering and leveraging biophysical drivers of membrane protein organization to design membrane systems could greatly enhance the functionality of these materials. Towards this goal, we use de novo protein design, molecular dynamic simulations, and cell-free systems to explore how membrane-protein hydrophobic mismatch could be used to tune protein cotranslational integration and organization in synthetic lipid membranes. We find that membranes must deform to accommodate membrane-protein hydrophobic mismatch, which reduces the expression and co-translational insertion of membrane proteins into synthetic membranes. We use this principle to sort proteins both between and within membranes, thereby achieving one-pot assembly of vesicles with distinct functions and controlled split-protein assembly, respectively. Our results shed light on protein organization in biological membranes and provide a framework to design self-organizing membrane-based materials with applications such as artificial cells, biosensors, and therapeutic nanoparticles.

Tuning of the Anti-Breast Cancer Activity of Betulinic Acid via Its Conversion to Ionic Liquids.

Betulinic acid (BA) is a natural pentacyclic triterpene with diverse biological activities. However, its low water solubility limits its pharmaceutical application. The conversion of pharmaceutically active molecules into ionic liquids (ILs) is a promising strategy to improve their physicochemical properties, stability, and/or potency. Here, we report the synthesis and characterization of 15 novel ILs containing a cation ethyl ester of a polar, non-polar, or charged amino acid [AAOEt] and an anion BA. Except for [ValOEt][BA], we observed preserved or up to 2-fold enhanced cytotoxicity toward hormone-dependent breast cancer cells MCF-7. The estimated IC50 (72 h) values within the series varied between 4.8 and 25.7 µM. We found that the most cytotoxic IL, [LysOEt][BA]2, reduced clonogenic efficiency to 20% compared to that of BA. In addition, we evaluated the effect of a 72 h treatment with BA or [LysOEt][BA]2, the most cytotoxic compound, on the thermodynamic behavior of MCF-7 cells. Based on our data, we suggest that the charged amino acid lysine included in the novel ILs provokes cytotoxicity by a mechanism involving alteration in membrane lipid organization, which could be accompanied by modulation of the visco-elastic properties of the cytoplasm.

Exploring the Physical Properties of Lipid Membranes with Polyhydroxy Oxanorbornane Head Group Using NBD-Conjugated and DPH Fluorescent Probes.

The present study focuses on exploring the physical properties of lipid membranes based on the polyhydroxy oxanorbornane (PH-ONB) headgroup, designed as synthetic analogues of naturally occurring archaeal lipid membranes. Specifically, we study two variants of PH-ONB headgroup-based lipids differing in the number of hydroxy groups present in the headgroup, with one having two hydroxy groups (ONB-2OH) and the other having three (ONB-3OH). These lipids form stable bilayer membranes. The study begins with a comprehensive analysis of the fluorescence characteristics of nitrobenzoxadiazole (NBD)-tagged ONB-based lipids in different solvent environments and within a model lipid membrane 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Subsequently, the physical properties of the ONB-based membranes were examined by using an NBD-tagged ONB-based probe and a commonly used extrinsic 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescent probe. The steady-state and time-resolved fluorescence properties of the NBD-tagged ONB-based probe and DPH were used to compare the physical properties of the ONB-based membranes, including polarity, fluidity, phase transition, order, hydration, location, heterogeneity, and rotational diffusion. The solid gel to liquid crystalline phase transition temperatures of ONB-2OH and ONB-3OH lipid membranes are found to be (68 ± 1) °C and (74 ± 1) °C, respectively. The variation in organization (size), fluidity, and phase transition temperature of ONB-based lipid membranes is explained by the extent of hydrogen bonding interactions between lipid head groups. ONB-based membranes exhibit characteristics similar to those of phospholipid membranes and possess a notably high phase transition temperature. These properties make them a promising and cost-effective synthetic alternative to archaeal lipid membranes with a wide range of potential applications.

Lipid Peroxidation Drives Liquid-Liquid Phase Separation and Disrupts Raft Protein Partitioning in Biological Membranes.

The peroxidation of membrane lipids by free radicals contributes to aging, numerous diseases, and ferroptosis, an iron-dependent form of cell death. Peroxidation changes the structure and physicochemical properties of lipids, leading to bilayer thinning, altered fluidity, and increased permeability of membranes in model systems. Whether and how lipid peroxidation impacts the lateral organization of proteins and lipids in biological membranes, however, remains poorly understood. Here, we employ cell-derived giant plasma membrane vesicles (GPMVs) as a model to investigate the impact of lipid peroxidation on ordered membrane domains, often termed membrane rafts. We show that lipid peroxidation induced by the Fenton reaction dramatically enhances the phase separation propensity of GPMVs into coexisting liquid-ordered (Lo) and liquid-disordered (Ld) domains and increases the relative abundance of the disordered phase. Peroxidation also leads to preferential accumulation of peroxidized lipids and 4-hydroxynonenal (4-HNE) adducts in the disordered phase, decreased lipid packing in both Lo and Ld domains, and translocation of multiple classes of raft proteins out of ordered domains. These findings indicate that the peroxidation of plasma membrane lipids disturbs many aspects of membrane rafts, including their stability, abundance, packing, and protein and lipid composition. We propose that these disruptions contribute to the pathological consequences of lipid peroxidation during aging and disease and thus serve as potential targets for therapeutic intervention.

Formation of EGFRwt/EGFRvIII homo- and hetero-dimers in glioblastoma cells as detected by single molecule localization microscopy.

Super-resolution microscopy has been used to show the formation of receptor clusters and adapted lipid organization of cell membranes for many members of the ErbB receptor family. The clustering behaviour depends on the receptor size and shape, possibly ligand binding or expression activity. Using single molecule localization microscopy (SMLM), we also showed this typical clustering for the epidermal growth factor receptor variant III (EGFRvIII) in glioblastoma multiforme (GBM) cells. EGFRvIII is co-expressed with the wild type (EGFRwt) and both receptors are assumed to preferentially form hetero-dimers leading to transactivation and elevated oncogenic EGFR-signalling in GBM cells. Here, we analysed EGFRvIII and EGFRwt co-localization using our already described model system of the glioblastoma cell line DKMG, displaying endogenous EGFRvIII expression. Using EGFRvIII and EGFRwt specific antibodies, EGFR localization and their potential for dimerization in a given membrane cluster were analysed by dual colour SMLM supported by novel approaches of mathematic evaluations including Ripley statistics, persistent homology and similarity algorithms. Surprisingly, cluster analysis, Ripley point-to-point distance statistics for cluster geometry and persistent homology comparing cluster topology, revealed that both EGFRvIII and EGFRwt do primarily not form hetero-dimers but the results support the hypothesis that they tend to form homo-dimers. The ratio of homo-dimers obtained by this calculation was significantly higher (>5σ, standard deviation) than expected from randomly arranged points. In comparison, hetero-dimer formation was only slightly increased. We confirmed these data by immunoprecipitation, which show no co-precipitation of EGFRvIII and EGFRwt. Furthermore, we showed that the topology of the clusters was more similar among the same type than among the different types of receptors. Taken together, these data indicate that EGFRvIII does induce oncogenic signalling by homo-dimerisation and not preferentially by hetero-dimer formation with EGFRwt. These data offer a new perspective on EGFRvIII signalling which will lead to a better understanding of this tumour associated receptor variant in GBM.

OSBP is a Major Determinant of Golgi Phosphatidylinositol 4-Phosphate Homeostasis.

The lipid phosphatidylinositol 4-phosphate (PI4P) plays a master regulatory role at Golgi membranes, orchestrating membrane budding, non-vesicular lipid transport and membrane organization. It follows that harmonious Golgi function requires strictly maintained PI4P homeostasis. One of the most abundant PI4P effector proteins is the oxysterol binding protein (OSBP), a lipid transfer protein that exchanges trans-Golgi PI4P for ER cholesterol. Although this protein consumes PI4P as part of its lipid anti-porter function, whether it actively contributes to Golgi PI4P homeostasis has been questioned. Here, we employed a series of acute and chronic genetic manipulations, together with orthogonal targeting of OSBP, to interrogate its control over Golgi PI4P abundance. Modulating OSBP levels at ER:Golgi membrane contact sites produces reciprocal changes in PI4P levels. Additionally, we observe that OSBP has a high capacity for PI4P turnover, even at orthogonal organelle membranes. However, despite also visiting the plasma membrane, endogenous OSBP makes no impact on PI4P levels in this compartment. We conclude that OSBP is a major determinant of Golgi PI4P homeostasis.

Structural Changes Induced by Resveratrol in Monounsaturated and Polyunsaturated Phosphatidylcholine-Enriched Model Membranes.

Resveratrol (Resv) is considered to exert a beneficial impact due to its radical scavenger, anti-microbial and anti-inflammatory properties through several mechanisms that could include its interaction with the cell plasma membrane. To address this issue, we investigated the influence of Resv on membrane lipid order and organization in large unilamellar vesicles composed of different lipids and ratios. The studied lipid membrane models were composed of phosphatidylcholine (PC) species (either palmitoyl-docosahexaenoyl phosphatidylcholine (PDPC) or palmitoyl-oleoyl phosphatidylcholine (POPC)), sphingomyelin (SM) and cholesterol (Chol). This study found that the addition of Resv resulted in complex membrane reorganization depending on the degree of fatty acid unsaturation at the sn-2 position, and the Lipid/Resv and SM/Chol ratios. Resv rigidified POPC-containing membranes and increased liquid-ordered (Lo) domain formation in 40/40/20 POPC/SM/Chol mixtures as this increase was lower at a 33/33/34 ratio. In contrast, Resv interacted with PDPC/SM/Chol mixtures in a bimodal manner by fluidizing/rigidifying the membranes in a dose-dependent way. Lo domain formation upon Resv addition occurred via the following bimodal mode of action: Lo domain size increased at low Resv concentrations; then, Lo domain size decreased at higher ones. To account for the variable effect of Resv, we suggest that it may act as a "spacer" at low doses, with a transition to a more "filler" position in the lipid bulk. We hypothesize that one of the roles of Resv is to tune the lipid order and organization of cell plasma membranes, which is closely linked to important cell functions such as membrane sorting and trafficking.

24S-Hydroxycholesterol in Neuropsychiatric Diseases: Schizophrenia, Autism Spectrum Disorder, and Bipolar Disorder.

Neuropsychiatric diseases (NPDs) are severe, debilitating psychiatric conditions that affect the nervous system. These are among the most challenging disorders in medicine. Some examples include Alzheimer's, anxiety disorders, autism spectrum disorder, bipolar disorder, and schizophrenia. NPDs represent an ever-increasing burden on public health and are prevalent throughout the world. For most of these diseases, the particular etiopathogeneses are still enigmatic. NPDs are also associated with structural and functional changes in the brain, along with altered neurotransmitter and neuroendocrine systems.Approximately 25% of the total human body cholesterol is located in the brain. Its involvement in neuronal functions starts in the early growth stages and remains important throughout adulthood. It is also an integral part of the neuronal membrane, ensuring membrane lipid organization and regulating membrane fluidity. The main mechanism for removing cholesterol from the brain is cholesterol 24-hydroxylation by cytochrome P450 46A1 (CYP46A1), an enzyme specifically found in the central nervous system. Although research on 24S-OHC and its role in neuropsychiatric diseases is still in its early stages, this oxidized cholesterol metabolite is thought to play a crucial role in the etiology of NPDs. 24S-OHC can affect neurons, astrocytes, oligodendrocytes, and vascular cells. In addition to regulating the homeostasis of cholesterol in the brain, this oxysterol is involved in neurotransmission, oxidative stress, and inflammation. The role of 24S-OHC in NPDs has been found to be controversial in terms of the findings so far. There are several intriguing discrepancies in the data gathered so far regarding 24S-OHC and NPDs. In fact, 24S-OHC levels were reported to have decreased in a number of NPDs and increased in others.Hence, in this chapter, we first summarize the available data regarding 24S-OHC as a biomarker in NPDs, including schizophrenia, autism spectrum disorder, and bipolar disorder. Then, we present a brief synopsis of the pharmacological targeting of 24S-OHC levels through the modulation of CYP46A1 activity.

Review of Eukaryote Cellular Membrane Lipid Composition, with Special Attention to the Fatty Acids.

Biological membranes, primarily composed of lipids, envelop each living cell. The intricate composition and organization of membrane lipids, including the variety of fatty acids they encompass, serve a dynamic role in sustaining cellular structural integrity and functionality. Typically, modifications in lipid composition coincide with consequential alterations in universally significant signaling pathways. Exploring the various fatty acids, which serve as the foundational building blocks of membrane lipids, provides crucial insights into the underlying mechanisms governing a myriad of cellular processes, such as membrane fluidity, protein trafficking, signal transduction, intercellular communication, and the etiology of certain metabolic disorders. Furthermore, comprehending how alterations in the lipid composition, especially concerning the fatty acid profile, either contribute to or prevent the onset of pathological conditions stands as a compelling area of research. Hence, this review aims to meticulously introduce the intricacies of membrane lipids and their constituent fatty acids in a healthy organism, thereby illuminating their remarkable diversity and profound influence on cellular function. Furthermore, this review aspires to highlight some potential therapeutic targets for various pathological conditions that may be ameliorated through dietary fatty acid supplements. The initial section of this review expounds on the eukaryotic biomembranes and their complex lipids. Subsequent sections provide insights into the synthesis, membrane incorporation, and distribution of fatty acids across various fractions of membrane lipids. The last section highlights the functional significance of membrane-associated fatty acids and their innate capacity to shape the various cellular physiological responses.

Dynamic Regulation of Order Lipid Meso-domains in the Capillary Endothelial Cell Membrane

The structure and function of ion channels are sensitive to direct interactions with lipids of the membrane and to the biophysical properties of the membrane microenvironment, such that membrane fluidity, lateral pressure profile, and thickness can modulate the channels. Cholesterol plays a significant role in regulating membrane fluidity and is capable of phase separation where cholesterol will spontaneously separate into its own domain, which is partially responsible for developing two distinct domains within the membrane. These ordered domains are regions of high cholesterol and low fluidity, and the disordered domains are regions of low cholesterol and high fluidity. Here we examine and provide evidence of the presence of ordered cholesterol membrane domains in capillary endothelial cells. We will test the hypothesis that capillary endothelial cell ordered domains function as a signaling hub for different phospholipids regulating ion channel function. Using the isolated retinal vasculature, confocal microscopy, and fluorescent Bodipy-cholesterol, a marker of ordered lipid domains, we observed large regions approximately 2.3± 0.9 μm long of cholesterol ordered meso-domains within capillary endothelial cells and not the feeding arteriole. To determine the dynamic nature of these meso-domains, we used membrane-disordering drugs to disrupt the organization of membrane lipids. The ordered meso-domains display dynamic membrane disordering by ethanol and can re-cluster into the large ordered meso-domains. In addition, we observed that the critical signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) co-localized with the ordered meso-domains. To determine the cooperativity between these lipid molecules, we examined the effects of ethanol-dependent disordering of cholesterol on the membrane localization of PIP2. In addition, we examined the effects of PIP2 depletion with the G-protein couple receptor agonist, U46619, on the disordered state of the cholesterol meso-domains. These data suggest that ordered meso-domains might play a role as a signaling hub affecting the membrane fluidity and bioavailability of signaling lipids modulating ion channel function in capillary endothelial cell. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer.

Hierarchical organization of integral membrane proteins (IMP) and lipids at the membrane is essential for regulating myriad downstream signaling. A quantitative understanding of these processes requires both detections of oligomeric organization of IMPs and lipids directly from intact membranes and determination of key membrane components and properties that regulate them. Addressing this, we have developed a platform that enables native mass spectrometry (nMS) analysis of IMP-lipid complexes directly from intact and customizable lipid membranes. Both the lipid composition and membrane properties (such as curvature, tension, and fluidity) of these bilayers can be precisely customized to a target membrane. Subsequent direct nMS analysis of these intact proteolipid vesicles can yield the oligomeric states of the embedded IMPs, identify bound lipids, and determine the membrane properties that can regulate the observed IMP-lipid organization. Applying this method, we show how lipid binding regulates neurotransmitter release and how membrane composition regulates the functional oligomeric state of a transporter.