Cyclo(Pro-Tyr) elicits conserved cellular damage in fungi by targeting the [H+]ATPase Pma1 in plasma membrane domains.

Bioactive metabolites play a crucial role in shaping interactions among diverse organisms. In this study, we identified cyclo(Pro-Tyr), a metabolite produced by Bacillus velezensis, as a potent inhibitor of Botrytis cinerea and Caenorhabditis elegans, two potential cohabitant eukaryotic organisms. Based on our investigation, cyclo(Pro-Tyr) disrupts plasma membrane polarization, induces oxidative stress and increases membrane fluidity, which compromises fungal membrane integrity. These cytological and physiological changes induced by cyclo(Pro-Tyr) may be triggered by the destabilization of membrane microdomains containing the [H+]ATPase Pma1. In response to cyclo(Pro-Tyr) stress, fungal cells activate a transcriptomic and metabolomic response, which primarily involves lipid metabolism and Reactive Oxygen Species (ROS) detoxification, to mitigate membrane damage. This similar response occurs in the nematode C. elegans, indicating that cyclo(Pro-Tyr) targets eukaryotic cellular membranes.

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.

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.

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.

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.

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.

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.

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.

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.