The biophysical drivers of membrane lateral heterogeneity, often termed lipid rafts, have been largely explored using synthetic liposomes or mammalian plasma membrane-derived giant vesicles. Yeast vacuoles, an organelle comparable to mammalian lysosomes, is the only in vivo system that shows stable micrometer scale phase separation in unperturbed cells. The ease of manipulating lipid metabolism in yeast makes this a powerful system for identifying lipids involved in the onset of vacuole membrane heterogeneity. Vacuole domains are induced by stationary stage growth and nutritional starvation, during which they serve as a docking and internalization site for lipid droplet energy stores. Here we describe methods for characterizing vacuole phase separation, its physiological function, and its lipidic drivers. First, we detail methodologies for robustly inducing vacuole domain formation and quantitatively characterizing during live cell imaging experiments. Second, we detail a new protocol for biochemical isolation of stationary stage vacuoles, which allows for lipidomic dissection of membrane phase separation. Third, we describe biochemical techniques for analyzing lipid droplet internalization in vacuole domains. When combined with genetic or chemical perturbations to lipid metabolism, these methods allow for systematic dissection of lipid composition in the structure and function of ordered membrane domains in living cells.

Membrane lipids and proteins form dynamic domains crucial for physiological and pathophysiological processes, including viral infection. Many plasma membrane proteins, residing within membrane domains enriched with cholesterol (CHOL) and sphingomyelin (SM), serve as receptors for attachment and entry of viruses into the host cell. Among these, human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), use proteins associated with membrane domains for initial binding and internalization. We hypothesized that the interaction of lipid-binding proteins with CHOL in plasma membrane could sequestrate lipids and thus affect the efficiency of virus entry into host cells, preventing the initial steps of viral infection. We have prepared CHOL-binding proteins with high affinities for lipids in the plasma membrane of mammalian cells. Binding of the perfringolysin O domain four (D4) and its variant D4E458L to membrane CHOL impaired the internalization of the receptor-binding domain of the SARS-CoV-2 spike protein and the pseudovirus complemented with the SARS-CoV-2 spike protein. SARS-CoV-2 replication in Vero E6 cells was also decreased. Overall, our results demonstrate that the integrity of CHOL-rich membrane domains and the accessibility of CHOL in the membrane play an essential role in SARS-CoV-2 cell entry.

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.

Lipid membrane nanodomains or lipid rafts are 10-200 nm diameter size cholesterol- and sphingolipid-enriched domains of the plasma membrane, gathering many proteins with different roles. Isolation and characterization of plasma membrane proteins by differential centrifugation and proteomic studies have revealed a remarkable diversity of proteins in these domains. The limited size of the lipid membrane nanodomain challenges the simple possibility that all of them can coexist within the same lipid membrane domain. As caveolin-1, flotillin isoforms and gangliosides are currently used as neuronal lipid membrane nanodomain markers, we first analyzed the structural features of these components forming nanodomains at the plasma membrane since they are relevant for building supramolecular complexes constituted by these molecular signatures. Among the proteins associated with neuronal lipid membrane nanodomains, there are a large number of proteins that play major roles in calcium signaling, such as ionotropic and metabotropic receptors for neurotransmitters, calcium channels, and calcium pumps. This review highlights a large variation between the calcium signaling proteins that have been reported to be associated with isolated caveolin-1 and flotillin-lipid membrane nanodomains. Since these calcium signaling proteins are scattered in different locations of the neuronal plasma membrane, i.e., in presynapses, postsynapses, axonal or dendritic trees, or in the neuronal soma, our analysis suggests that different lipid membrane-domain subtypes should exist in neurons. Furthermore, we conclude that classification of lipid membrane domains by their content in calcium signaling proteins sheds light on the roles of these domains for neuronal activities that are dependent upon the intracellular calcium concentration. Some examples described in this review include the synaptic and metabolic activity, secretion of neurotransmitters and neuromodulators, neuronal excitability (long-term potentiation and long-term depression), axonal and dendritic growth but also neuronal cell survival and death.

Molecular oxygen (O2) is the perfect probe molecule for membrane studies carried out using the saturation recovery EPR technique. O2 is a small, paramagnetic, hydrophobic enough molecule that easily partitions into a membrane\'s different phases and domains. In membrane studies, the saturation recovery EPR method requires two paramagnetic probes: a lipid-analog nitroxide spin label and an oxygen molecule. The experimentally derived parameters of this method are the spin-lattice relaxation times (T 1s) of spin labels and rates of bimolecular collisions between O2 and the nitroxide fragment. Thanks to the long T 1 of lipid spin labels (from 1 to 10 μs), the approach is very sensitive to changes of the local (around the nitroxide fragment) O2 diffusion-concentration product. Small variations in the lipid packing affect O2 solubility and O2 diffusion, which can be detected by the shortening of T 1 of spin labels. Using O2 as a probe molecule and a different lipid spin label inserted into specific phases of the membrane and membrane domains allows data about the lateral arrangement of lipid membranes to be obtained. Moreover, using a lipid spin label with the nitroxide fragment attached to its head group or a hydrocarbon chain at different positions also enables data about molecular dynamics and structure at different membrane depths to be obtained. Thus, the method can be used to investigate not only the lateral organization of the membrane (i.e., the presence of membrane domains and phases), but also the depth-dependent membrane structure and dynamics, and, hence, the membrane properties in three dimensions.

The continuous wave EPR spin-labeling method was used to evaluate age-related changes in the amounts of phospholipids (PLs) and cholesterol (Chol) in domains present in intact, cortical, and nuclear fiber cell plasma membranes isolated separately from the left and right eye lenses of the same human donor. The relative amounts of boundary plus trapped PLs were evaluated with the PL analog 12-doxylstearic acid spin label (12-SASL) and the relative amounts of trapped Chol with the Chol analog androstane spin label (ASL). The donors ranged in age from 15 to 70 years. Both the left and right eye lenses from donors aged 60, 65, and 70 years had nuclear cataracts; additionally, the right eye lens only of the 60-year-old donor had a cortical cataract. In transparent lenses, the relative amounts of boundary plus trapped PLs increase monotonously with donor age, and, at all ages, this amount was greater in nuclear compared with cortical membranes. Moreover, in transparent lenses, the relative amount of trapped Chol increases with age in nuclear membranes. However, the EPR spectrum of ASL from cortical membranes of 15- to 60-year-old donors shows only the weakly immobilized component assigned to ASL in the bulk plus Chol bilayer domain. Only the cortical membranes of 61- to 70-year-old donors contain both weakly and strongly immobilized components. The strongly immobilized component is assigned to ASL in trapped lipids. We speculate that the age of 60 years may be considered as a \"threshold\" for appearance of trapped lipids in cortical membranes. The relative amounts of boundary plus trapped PLs in lenses with nuclear cataracts is lower than that predicted from the tendency of the age-dependent increase observed for transparent lenses. The differences in amounts of lipids in the indicated left and right eye domains of each donor are smaller than the differences in single donors of a similar age.

The Fluid-Mosaic Membrane (FMM) model was originally proposed as a general, nanometer-scale representation of cell membranes (Singer and Nicolson, 1972). The FMM model was based on some general principles, such as thermodynamic considerations, intercalation of globular proteins into a lipid bilayer, independent protein and lipid dynamics, cooperativity and other characteristics. Other models had trimolecular structures or membrane globular lipoprotein units. These latter models were flawed, because they did not allow autonomous lipids, membrane domains or discrete lateral dynamics. The FMM model was also consistent with membrane asymmetry, cis- and trans-membrane linkages and associations of membrane components into multi-molecular complexes and domains. It has remained useful for explaining the basic organizational principles and properties of various biological membranes. New information has been added, such as membrane-associated cytoskeletal assemblies, extracellular matrix interactions, transmembrane controls, specialized lipid-protein domains that differ in compositions, rotational and lateral mobilities, lifetimes, functions, and other characteristics. The presence of dense, structured membrane domains has reduced significantly the extent of fluid-lipid membrane areas, and the FMM model is now considered to be more mosaic and dense than the original proposal.

The plasma membrane is a complex assembly of proteins and lipids that can self-assemble in submicroscopic domains commonly termed \"lipid rafts\", which are implicated in membrane signaling and trafficking. Recently, photo-sensitive lipids were introduced to study membrane domain organization, and photo-isomerization was shown to trigger the mixing and de-mixing of liquid-ordered (lo ) domains in artificial phase-separated membranes. Here, we synthesized globotriaosylceramide (Gb3 ) glycosphingolipids that harbor an azobenzene moiety at different positions of the fatty acid to investigate light-induced membrane domain reorganization, and that serve as specific receptors for the protein Shiga toxin (STx). Using phase-separated supported lipid bilayers on mica surfaces doped with four different photo-Gb3 molecules, we found by fluorescence microscopy and atomic force microscopy that liquid disordered (ld ) domains were formed within lo domains upon trans-cis photo-isomerization. The fraction and size of these ld domains were largest for Gb3 molecules with the azobenzene group at the end of the fatty acid. We further investigated the impact of domain reorganization on the interaction of the B-subunits of STx with the photo-Gb3 . Fluorescence and atomic force micrographs clearly demonstrated that STxB binds to the lo phase if Gb3 is in the trans-configuration, whereas two STxB populations are formed if the photo-Gb3 is switched to the cis-configuration highlighting the idea of manipulating lipid-protein interactions with a light stimulus.

Compartmentalization, together with transbilayer and lateral asymmetries, provide the structural foundation for functional specializations at the cell surface, including the active role of the lipid microenvironment in the modulation of membrane-bound proteins. The chemical synapse, the site where neurotransmitter-coded signals are decoded by neurotransmitter receptors, adds another layer of complexity to the plasma membrane architectural intricacy, mainly due to the need to accommodate a sizeable number of molecules in a minute subcellular compartment with dimensions barely reaching the micrometer. In this review, we discuss how nature has developed suitable adjustments to accommodate different types of membrane-bound receptors and scaffolding proteins via membrane microdomains, and how this \"effort-sharing\" mechanism has evolved to optimize crosstalk, separation, or coupling, where/when appropriate. We focus on a fast ligand-gated neurotransmitter receptor, the nicotinic acetylcholine receptor, and a second-messenger G-protein coupled receptor, the cannabinoid receptor, as a paradigmatic example.

The Fluid-Mosaic Model has been the accepted general or basic model for biomembrane structure and organization for the last 50 years. In order to establish a basic model for biomembranes, some general principles had to be established, such as thermodynamic assumptions, various molecular interactions, component dynamics, macromolecular organization and other features. Previous researchers placed most membrane proteins on the exterior and interior surfaces of lipid bilayers to form trimolecular structures or as lipoprotein units arranged as modular sheets. Such membrane models were structurally and thermodynamically unsound and did not allow independent lipid and protein lateral movements. The Fluid-Mosaic Membrane Model was the only model that accounted for these and other characteristics, such as membrane asymmetry, variable lateral movements of membrane components, cis- and transmembrane linkages and dynamic associations of membrane components into multimolecular complexes. The original version of the Fluid-Mosaic Membrane Model was never proposed as the ultimate molecular description of all biomembranes, but it did provide a basic framework for nanometer-scale biomembrane organization and dynamics. Because this model was based on available 1960s-era data, it could not explain all of the properties of various biomembranes discovered in subsequent years. However, the fundamental organizational and dynamic aspects of this model remain relevant to this day. After the first generation of this model was published, additional data on various structures associated with membranes were included, resulting in the addition of membrane-associated cytoskeletal, extracellular matrix and other structures, specialized lipid-lipid and lipid-protein domains, and other configurations that can affect membrane dynamics. The presence of such specialized membrane domains has significantly reduced the extent of the fluid lipid membrane matrix as first proposed, and biomembranes are now considered to be less fluid and more mosaic with some fluid areas, rather than a fluid matrix with predominantly mobile components. However, the fluid-lipid matrix regions remain very important in biomembranes, especially those involved in the binding and release of membrane lipid vesicles and the uptake of various nutrients. Membrane phospholipids can associate spontaneously to form lipid structures and vesicles that can fuse with various cellular membranes to transport lipids and other nutrients into cells and organelles and expel damaged lipids and toxic hydrophobic molecules from cells and tissues. This process and the clinical use of membrane phospholipid supplements has important implications for chronic illnesses and the support of healthy mitochondria, plasma membranes and other cellular membrane structures.