Structure and dynamics of cholesterol-mediated aquaporin-0 arrays and implications for lipid rafts.

Aquaporin-0 (AQP0) tetramers form square arrays in lens membranes through a yet unknown mechanism, but lens membranes are enriched in sphingomyelin and cholesterol. Here, we determined electron crystallographic structures of AQP0 in sphingomyelin/cholesterol membranes and performed molecular dynamics (MD) simulations to establish that the observed cholesterol positions represent those seen around an isolated AQP0 tetramer and that the AQP0 tetramer largely defines the location and orientation of most of its associated cholesterol molecules. At a high concentration, cholesterol increases the hydrophobic thickness of the annular lipid shell around AQP0 tetramers, which may thus cluster to mitigate the resulting hydrophobic mismatch. Moreover, neighboring AQP0 tetramers sandwich a cholesterol deep in the center of the membrane. MD simulations show that the association of two AQP0 tetramers is necessary to maintain the deep cholesterol in its position and that the deep cholesterol increases the force required to laterally detach two AQP0 tetramers, not only due to protein-protein contacts but also due to increased lipid-protein complementarity. Since each tetramer interacts with four such 'glue' cholesterols, avidity effects may stabilize larger arrays. The principles proposed to drive AQP0 array formation could also underlie protein clustering in lipid rafts.

ASMase is Essential for the Immune Response to Partial-Tumor Radiation Exposure.

BACKGROUND/AIMS: Tumor response to radiation is thought to depend on the direct killing of tumor cells. Our laboratory has called this into question. Firstly, we showed that the biology of the host, specifically the endothelial expression of acid sphingomyelinase (ASMase), was critical in determining tumor radiocurability. Secondly, we have shown that the immune system can enhance radiation response by allowing a complete tumor control in hemi-irradiated tumors. In this paper, we focus on the integration of these two findings. METHODS: We used Lewis Lung Carcinoma (LLC) cells, injected in the flank of either: (i) ASMase knockout or (ii) WT of matched background (sv129xBl/6) or (iii) C57Bl/6 mice. Radiation therapy (RT) was delivered to 50% or 100% of the LLC tumor volume. Tumor response, immune infiltration (CD8+ T cells), ICAM-1, and STING activation were measured. Radiotherapy was also combined with methyl-cyclodextrin, to inhibit the ASMase-mediated formation of ceramide-enriched lipid rafts. RESULTS: We recapitulated our previous finding, namely that tumor hemi-irradiation was sufficient for tumor control in the LLC/C57Bl/6 model. However, in ASMase KO mice hemi-irradiation was ineffective. Likewise, pharmacological inhibition of ASMase significantly reduced the tumor response to hemi-irradiation. Further, we demonstrated elevated ICAM-1 expression, increased levels of CD8+ T cells, ICAM-1, and STING activation in tumors growing in C57Bl/6 mice, as well as the ASMase WT strain. However, no such changes were seen in tumors growing in ASMase KO mice. CONCLUSION: ASMase and ceramide generation are necessary to mediate a radiation-induced anti-tumor immune response via STING activation.

Biomimetic Lipid Raft: Domain Stability and Interaction with Physiologically Active Molecules.

The cell membrane, also called the plasma membrane, is the membrane on the cytoplasmic surface that separates the extracellular from the intracellular. It is thin, about 10 nm thick when viewed with an electron microscope, and is composed of two monolayers of phospholipid membranes (lipid bilayers) containing many types of proteins. It is now known that this cell membrane not only separates the extracellular from the intracellular, but is also involved in sensory stimuli such as pain, itching, sedation, and excitement. Since the "Fluid mosaic model" was proposed for cell membranes, molecules have been thought to be homogeneously distributed on the membrane surface. Later, at the end of the twentieth century, the existence of "Phase-separated microdomain structures" consisting of ordered phases rich in saturated lipids and cholesterol was suggested, and these were termed "Lipid rafts." A model in which lipid rafts regulate cell signaling has been proposed and is the subject of active research.This chapter first outlines the physicochemical properties and thermodynamic models of membrane phase separation (lipid rafts), which play an important role in cell signaling. Next, how physiologically active molecules such as local anesthetics, cooling agents (menthol), and warming agents (capsaicin) interact with artificial cell membranes will be presented.It is undeniable that the plasma membrane contains many channels and receptors that are involved in the propagation of sensory stimuli. At the same time, however, it is important to understand that the membrane exerts a significant influence on the intensity and propagation of these stimuli.

ACSL4-mediated lipid rafts prevent membrane rupture and inhibit immunogenic cell death in melanoma.

Chemotherapy including platinum-based drugs are a possible strategy to enhance the immune response in advanced melanoma patients who are resistant to immune checkpoint blockade (ICB) therapy. However, the immune-boosting effects of these drugs are a subject of controversy, and their impact on the tumor microenvironment are poorly understood. In this study, we discovered that lipid peroxidation (LPO) promotes the formation of lipid rafts in the membrane, which mediated by Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) impairs the sensitivity of melanoma cells to platinum-based drugs. This reduction primarily occurs through the inhibition of immunogenic ferroptosis and pyroptosis by reducing cell membrane pore formation. By disrupting ACSL4-mediaged lipid rafts via the removal of membrane cholesterol, we promoted immunogenic cell death, transformed the immunosuppressive environment, and improved the antitumor effectiveness of platinum-based drugs and immune response. This disruption also helped reverse the decrease in CD8+ T cells while maintaining their ability to secrete cytokines. Our results reveal that ACSL4-dependent LPO is a key regulator of lipid rafts formation and antitumor immunity, and that disrupting lipid rafts has the potential to enhance platinum-based drug-induced immunogenic ferroptosis and pyroptosis in melanoma. This novel strategy may augment the antitumor immunity of platinum-based therapy and further complement ICB therapy.

Zdhhc1- and Zdhhc2-mediated Gpm6a palmitoylation is essential for maintenance of mammary stem cell activity.

Adult mammary stem cells (aMaSCs) are vital to tissue expansion and remodeling during the process of postnatal mammary development. The protein C receptor (Procr) is one of the well-identified surface markers of multipotent aMaSCs. However, an understanding of the regulatory mechanisms governing Procr's protein stability remains incomplete. In this study, we identified Glycoprotein m6a (Gpm6a) as a critical protein for aMaSC activity modulation by using the Gpm6a knockout mouse model. Interestingly, we determined that Gpm6a depletion results in a reduction of Procr protein stability. Mechanistically, Gpm6a regulates Procr protein stability by mediating the formation of lipid rafts, a process requiring Zdhhc1 and Zdhhc2 to palmitate Gpm6a at Cys17,18 and Cys246 sites. Our findings highlight an important mechanism involving Zdhhc1- and Zdhhc2-mediated Gpm6a palmitoylation for the regulation of Procr stability, aMaSC activity, and postnatal mammary development.

Dynamic swarms regulate the morphology and distribution of soft membrane domains.

We study the dynamic structure of lipid domain inclusions embedded within a phase-separated reconstituted lipid bilayer in contact with a swarming flow of gliding filamentous actin. Passive circular domains transition into highly deformed morphologies that continuously elongate, rotate, and pinch off into smaller fragments, leading to a dynamic steady state with ≈23× speedup in the relaxation of the intermediate scattering function compared with passive membrane domains driven by purely thermal forces. To corroborate experimental results, we develop a phase-field model of the lipid domains with two-way coupling to the Toner-Tu equations. We report phase domains that become entrained in the chaotic eddy patterns, with oscillating waves of domains that correlate with the dominant wavelengths of the Toner-Tu flow fields.

Tensing Flipper: Photosensitized Manipulation of Membrane Tension, Lipid Phase Separation, and Raft Protein Sorting in Biological Membranes.

The lateral organization of proteins and lipids in the plasma membrane is fundamental to regulating a wide range of cellular processes. Compartmentalized ordered membrane domains enriched with specific lipids, often termed lipid rafts, have been shown to modulate the physicochemical and mechanical properties of membranes and to drive protein sorting. Novel methods and tools enabling the visualization, characterization, and/or manipulation of membrane compartmentalization are crucial to link the properties of the membrane with cell functions. Flipper, a commercially available fluorescent membrane tension probe, has become a reference tool for quantitative membrane tension studies in living cells. Here, we report on a so far unidentified property of Flipper, namely, its ability to photosensitize singlet oxygen (1O2) under blue light when embedded into lipid membranes. This in turn results in the production of lipid hydroperoxides that increase membrane tension and trigger phase separation. In biological membranes, the photoinduced segregated domains retain the sorting ability of intact phase-separated membranes, directing raft and nonraft proteins into ordered and disordered regions, respectively, in contrast to radical-based photo-oxidation reactions that disrupt raft protein partitioning. The dual tension reporting and photosensitizing abilities of Flipper enable simultaneous visualization and manipulation of the mechanical properties and lateral organization of membranes, providing a powerful tool to optically control lipid raft formation and to explore the interplay between membrane biophysics and cell function.

Guidelines for naming and studying plasma membrane domains in plants.

Biological membranes play a crucial role in actively hosting, modulating and coordinating a wide range of molecular events essential for cellular function. Membranes are organized into diverse domains giving rise to dynamic molecular patchworks. However, the very definition of membrane domains has been the subject of continuous debate. For example, in the plant field, membrane domains are often referred to as nanodomains, nanoclusters, microdomains, lipid rafts, membrane rafts, signalling platforms, foci or liquid-ordered membranes without any clear rationale. In the context of plant-microbe interactions, microdomains have sometimes been used to refer to the large area at the plant-microbe interface. Some of these terms have partially overlapping meanings at best, but they are often used interchangeably in the literature. This situation generates much confusion and limits conceptual progress. There is thus an urgent need for us as a scientific community to resolve these semantic and conceptual controversies by defining an unambiguous nomenclature of membrane domains. In this Review, experts in the field get together to provide explicit definitions of plasma membrane domains in plant systems and experimental guidelines for their study. We propose that plasma membrane domains should not be considered on the basis of their size alone but rather according to the biological system being considered, such as the local membrane environment or the entire cell.

Polar Glycerolipids and Membrane Lipid Rafts.

Current understanding of the structure and functioning of biomembranes is impossible without determining the mechanism of formation of membrane lipid rafts. The formation of liquid-ordered and disordered phases (Lo and Ld) and lipid rafts in membranes and their simplified models is discussed. A new consideration of the processes of formation of lipid phases Lo and Ld and lipid rafts is proposed, taking into account the division of each of the glycerophospholipids into several groups. Generally accepted three-component schemes for modeling the membrane structure are critically considered. A four-component scheme is proposed, which is designed to more accurately assume the composition of lipids in the resulting Lo and Ld phases. The role of the polar head groups of phospholipids and, in particular, phosphatidylethanolamine is considered. The structure of membrane rafts and the possible absence of a clear boundary between the Lo and Ld phases are discussed.

Flotillin-mediated stabilization of unfolded proteins in bacterial membrane microdomains.

The function of many bacterial processes depends on the formation of functional membrane microdomains (FMMs), which resemble the lipid rafts of eukaryotic cells. However, the mechanism and the biological function of these membrane microdomains remain unclear. Here, we show that FMMs in the pathogen methicillin-resistant Staphylococcus aureus (MRSA) are dedicated to confining and stabilizing proteins unfolded due to cellular stress. The FMM scaffold protein flotillin forms a clamp-shaped oligomer that holds unfolded proteins, stabilizing them and favoring their correct folding. This process does not impose a direct energy cost on the cell and is crucial to survival of ATP-depleted bacteria, and thus to pathogenesis. Consequently, FMM disassembling causes the accumulation of unfolded proteins, which compromise MRSA viability during infection and cause penicillin re-sensitization due to PBP2a unfolding. Thus, our results indicate that FMMs mediate ATP-independent stabilization of unfolded proteins, which is essential for bacterial viability during infection.

Using lipid binding proteins and advanced microscopy to study lipid domains.

Sphingomyelin is postulated to form clusters with glycosphingolipids, cholesterol and other sphingomyelin molecules in biomembranes through hydrophobic interaction and hydrogen bonds. These clusters form submicron size lipid domains. Proteins that selectively binds sphingomyelin and/or cholesterol are useful to visualize the lipid domains. Due to their small size, visualization of lipid domains requires advanced microscopy techniques in addition to lipid binding proteins. This Chapter describes the method to characterize plasma membrane sphingomyelin-rich and cholesterol-rich lipid domains by quantitative microscopy. This Chapter also compares different permeabilization methods to visualize intracellular lipid domains.

Applications of phase-separating multi-bilayers in protein-membrane domain interactions.

Synthetic model membranes are important tools to elucidate lipid domain and protein interactions due to predefined lipid compositions and characterizable biophysical properties. Here, we introduce a model membrane with multiple lipid bilayers (multi-bilayers) stacked on a mica substrate that is prepared through a spin-coating technique. The spin-coated multi-bilayers are useful in the study of phase separated membranes with a high cholesterol content, mobile lipids, microscopic and reversible phase separation, and easy conjugation with proteins, which make them a good model to study interactions between proteins and membrane domains.

Using the yeast vacuole as a system to test the lipidic drivers of membrane heterogeneity in living cells.

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.

Pore-spanning membranes as a tool to investigate lateral lipid membrane heterogeneity.

Over the years, it has become more and more obvious that lipid membranes show a very complex behavior. This behavior arises in part from the large number of different kinds of lipids and proteins and how they dynamically interact with each other. In vitro studies using artificial membrane systems have shed light on the heterogeneity based on lipid-lipid interactions in multicomponent bilayer mixtures. Inspired by the raft hypothesis, the coexistence of liquid-disordered (ld) and liquid-ordered (lo) phases has drawn much attention. It was shown that ternary lipid mixtures containing low- and high-melting temperature lipids and cholesterol can phase separate into a lo phase enriched in the high-melting lipids and cholesterol and a ld phase enriched in the low-melting lipids. Depending on the model membrane system under investigation, different domain sizes, shapes, and mobilities have been found. Here, we describe how to generate phase-separated lo/ld phases in model membrane systems termed pore-spanning membranes (PSMs). These PSMs are prepared on porous silicon substrates with pore sizes in the micrometer regime. A proper functionalization of the top surface of the substrates is required to achieve the spreading of giant unilamellar vesicles (GUVs) to obtain PSMs. Starting with lo/ld phase-separated GUVs lead to membrane heterogeneities in the PSMs. Depending on the functionalization strategy of the top surface of the silicon substrate, different membrane heterogeneities are observed in the PSMs employing fluorescence microscopy. A quantitative analysis of the heterogeneity as well as the dynamics of the lipid domains is described.

Enhancing extracellular vesicle cargo loading and functional delivery by engineering protein-lipid interactions.

Naturally generated lipid nanoparticles termed extracellular vesicles (EVs) hold significant promise as engineerable therapeutic delivery vehicles. However, active loading of protein cargo into EVs in a manner that is useful for delivery remains a challenge. Here, we demonstrate that by rationally designing proteins to traffic to the plasma membrane and associate with lipid rafts, we can enhance loading of protein cargo into EVs for a set of structurally diverse transmembrane and peripheral membrane proteins. We then demonstrate the capacity of select lipid tags to mediate increased EV loading and functional delivery of an engineered transcription factor to modulate gene expression in target cells. We envision that this technology could be leveraged to develop new EV-based therapeutics that deliver a wide array of macromolecular cargo.

Phase separation in model lipid membranes investigated with cryogenic electron microscopy.

We describe a method for investigating lateral membrane heterogeneity using cryogenic electron microscopy (cryo-EM) images of liposomes. The method takes advantage of differences in the thickness and molecular density of ordered and disordered phases that are resolvable in phase contrast cryo-EM. Compared to biophysical techniques like FRET or neutron scattering that yield ensemble-averaged information, cryo-EM provides direct visualization of individual vesicles and can therefore reveal variability that would otherwise be obscured by averaging. Moreover, because the contrast mechanism involves inherent properties of the lipid phases themselves, no extrinsic probes are required. We explain and discuss various complementary analyses of spatially resolved thickness and intensity measurements that enable an assessment of the membrane's phase state. The method opens a window to nanodomain structure in synthetic and biological membranes that should lead to an improved understanding of lipid raft phenomena.

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.

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.

Membrane-bound Heat Shock Protein mHsp70 Is Required for Migration and Invasion of Brain Tumors.

Molecular chaperones, especially 70 kDa heat shock protein, in addition to their intracellular localization in cancer cells, can be exposed on the surface of the plasma membrane. We report that the membrane-associated chaperone mHsp70 of malignant brain tumors is required for high migratory and invasive activity of cancer cells. Live-cell inverted confocal microscopy of tumor samples from adult (n = 23) and pediatric (n = 9) neurooncologic patients showed pronounced protein expression on the membrane, especially in the perifocal zone. Mass spectrometry analysis of lipid rafts isolated from tumor cells confirmed the presence of the protein in the chaperone cluster (including representatives of other families, such as Hsp70, Hsc70, Hsp105, and Hsp90), which in turn, during interactome analysis, was associated with proteins involved in cell migration (e.g., Rac1, RhoC, and myosin-9). The use of small-molecule inhibitors of HSP70 (PES and JG98) led to a substantial decrease in the invasive potential of cells isolated from a tumor sample of patients, which indicates the role of the chaperone in invasion. Moreover, the use of HSP70 inhibitors in animal models of orthotopic brain tumors significantly delayed tumor progression, which was accompanied by an increase in overall survival. Data demonstrate that chaperone inhibitors, particularly JG98, disrupt the function of mHsp70, thereby providing an opportunity to better understand the diverse functions of this protein and offer aid in the development of novel cancer therapies. SIGNIFICANCE: Membrane-bound mHsp70 is required for brain tumor cell migration and invasion and therefore could be employed as a target for anticancer therapies.

How Membrane Phospholipids Containing Long-Chain Polyunsaturated Fatty Acids and Their Oxidation Products Orchestrate Lipid Raft Dynamics to Control Inflammation.

BACKGROUND: Long-chain PUFA (LC-PUFA) influence varying aspects of inflammation. One mechanism by which they regulate inflammation is by controlling the size and molecular composition of lipid rafts. Lipid rafts are sphingolipid/cholesterol-enriched plasma membrane microdomains that compartmentalize signaling proteins and thereby control downstream inflammatory gene expression and cytokine production. OBJECTIVES: This review summarizes developments in our understanding of how LC-PUFA acyl chains of phospholipids, in addition to oxidized derivatives of LC-PUFAs such as oxidized 1-palmitoyl-2-arachidonyl-phosphatidylcholine (oxPAPC), manipulate formation of lipid rafts and thereby inflammation. METHODS: We reviewed the literature, largely from the past 2 decades, on the impact of LC-PUFA acyl chains and oxidized products of LC-PUFAs on lipid raft biophysical organization of myeloid and lymphoid cells. The majority of the studies are based on rodent or cellular experiments with supporting mechanistic studies using biomimetic membranes and molecular dynamic simulations. These studies have focused largely on the LC-PUFA docosahexaenoic acid, with some studies addressing eicosapentaenoic acid. A few studies have investigated the role of oxidized phospholipids on rafts. RESULTS: The biophysical literature suggests a model in which n-3 LC-PUFAs, in addition to oxPAPC, localize predominately to nonraft regions and impart a disordering effect in this environment. Rafts become larger because of the ensuing increase in the difference in order between raft and nonrafts. Biochemical studies suggest that some n-3 LC-PUFAs can be found within rafts. This deviation from homeostasis is a potential trigger for controlling aspects of innate and adaptive immunity. CONCLUSION: Overall, select LC-PUFA acyl chains and oxidized acyl chains of phospholipids control lipid raft dynamics and downstream inflammation. Gaps in knowledge remain, particularly on underlying molecular mechanisms by which plasma membrane receptor organization is controlled in response to oxidized LC-PUFA acyl chains of membrane phospholipids. Validation in humans is also an area for future study.