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Plasma membrane (PM) nanodomains have emerged as pivotal elements in the regulation of plant cellular functions and signal transduction. These nanoscale membrane regions, enriched in specific lipids and proteins, behave as regulatory/signaling hubs spatially and temporally coordinating critical cellular functions. In this review, we first examine the mechanisms underlying the formation and maintenance of PM nanodomains in plant cells, highlighting the roles of PM lipid composition, protein oligomerization and interactions with cytoskeletal and cell wall components. Then, we discuss how nanodomains act as organizing centers by mediating protein-protein interactions that orchestrate essential processes such as symbiosis, defense against pathogens, ion transport or hormonal and reactive oxygen species (ROS) signaling. Finally, we introduce the concept of nanoenvironments, where localized physicochemical variations are generated in the very close proximity of PM nanodomains, in response to stimuli. After decoding by a dedicated machinery likely localized in the vicinity of nanodomains, this enrichment of secondary messengers, such as ROS or Ca 2+ , would allow specific downstream cellular responses. This review provides insights into the dynamic nature of nanodomains and proposes future research to better understand their contribution to the intricate signaling networks that govern plant development and stress responses., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Membrane fusion occurs at the early stages of SARS-CoV-2 replication, during entry of the virus, and later during the formation of multinucleated cells called syncytia. Fusion is mediated by the binding of the viral Spike protein to its receptor ACE2. Lipid rafts are dynamic nanodomains enriched in cholesterol and sphingolipids. Rafts can act as platforms for entry of different viruses by localizing virus receptors, and attachment factors to the same membrane domains. Here, we first demonstrate that cholesterol depletion by methyl-beta-cyclodextrin inhibits Spike-mediated fusion and entry. To further study the role of ACE2 lipid raft localization in SARS-CoV-2 fusion and entry, we designed a GPI-anchored ACE2 construct. Both ACE2 and ACE2-GPI proteins were similarly expressed at the plasma membrane. Through membrane flotation assays, we show that in different cell lines, ACE2-GPI localizes predominantly to raft domains of the plasma membrane while ACE2 is non-raft associated. We then compare the ability of ACE2 and ACE2-GPI to permit SARS-CoV-2 entry, replication, and syncytia formation of different viral variants. We find little difference in the two proteins. Our results demonstrate that SARS-CoV-2 entry and fusion are cholesterol-dependent and raft-independent processes.IMPORTANCERafts are often exploited by viruses and used as platforms to enhance their entry into the cell or spread from cell to cell. The membrane localization of ACE2 and the role of lipid rafts in SARS-CoV-2 entry and cell-to-cell spread are poorly understood. The function of lipid rafts in viral fusion is often studied through their disruption by cholesterol-depleting agents. However, this process may have off-target impacts on viral fusion independently of lipid-raft disruption. Therefore, we created an ACE2 construct that localizes to lipid rafts using a GPI anchor. Conversely, wild-type ACE2 was non-raft associated. We find that the localization of ACE2 to lipid rafts does not modify the fusion dynamics of SARS-CoV-2., Competing Interests: The authors declare no conflict of interest.

Solvent toxicity limits n -butanol fermentation titer, increasing the cost and energy consumption for subsequent separation processes and making biobased production more expensive and energy-intensive than petrochemical approaches. Amphiphilic solvents such as n -butanol partition into the cell membrane of fermenting microorganisms, thinning the transverse structure, and eventually causing a loss of membrane potential and cell death. In this work, we demonstrate the deleterious effects of n -butanol partitioning upon the lateral dimension of the membrane structure, called membrane domains or lipid rafts. Lipid rafts are regions of the cell membrane enriched with certain lipids, providing a reservoir of high melting temperature lipids and a platform for membrane protein partitioning and oligomerization. Neutron scattering experiments and molecular dynamics simulations revealed that n -butanol increased the size of the lipid domains in a model membrane system. The data showed that n -butanol partitions more into the disordered lipid regions than into the raft-like phase, leading to a differential thinning of these coexisting phases in the plane of the membrane and increasing the hydrophobic mismatch. The resulting increase in line tension at the interface favors domain coalescence to minimize the ratio of the interfacial length to domain area. A detailed computational investigation of the lipid domain interface identifies the boundary as a site of membrane disorder and thinning due to an accumulation of n -butanol. Solvent-induced changes to domain morphology and membrane instability at the domain interface are unrecognized modes of solvent-induced stress to fermenting microbes, representing targets for new solvent tolerance strategies to increase the n -butanol titer.

Lipid rafts are liquid-ordered domains in which specific enzymes and receptors are located. These membrane platforms play crucial roles in a variety of signaling pathways. Alterations in the lipid environment, such as those elicited by oxidative stress, can lead to important functional disruptions in membrane proteins. Cell membrane microarrays have emerged in the past decade as a powerful methodology for the study of both lipids and membrane proteins at large scales. Based on that technology and the importance of liquid-ordered subdomains, we have developed a new printed lipid raft technology with a preserved native protein structure and lipid environment. To validate this technology and evaluate its potential for different aims, raft membrane microarrays (RMMAs) containing two different cell types (astrocytes and neurons) and three different conditions (astrocytes in control situation, metabolic stress, and oxidative stress) were developed. To study differences in lipid profiles between raft domains, the MALDI-MS assay was performed on RMMAs. To evaluate the preservation of native protein activities (enzymatic activity and ligand binding) in the printed raft domains, differences in NADH oxidoreductase, GAPDH, cholinesterase activities, and sigma-1 and sigma-2 binding assays were performed. We demonstrate the performance of this new microarray technology, adapted to membrane subdomains, as valid to explore changes in lipid composition and protein activities in raft domains from brain cell lines under different stress conditions relevant for neuropathology.

Chimeric antigen receptor T cells (CART) targeting CD19 through CD28.ζ signaling induce rapid lysis of leukemic blasts, contrasting with persistent tumor control exhibited by 4-1BB.ζ-CART. We reasoned that molecular dynamics at the CART immune synapse (CARIS) could explain differences in their tumor rejection kinetics. We observed that CD28.ζ-CART engaged in brief highly lethal CARIS and mastered serial killing, whereas 4-1BB.ζ-CART formed lengthy CARIS and relied on robust expansion and cooperative killing. We analyzed CARIS membrane lipid rafts (mLRs) and found that, upon tumor engagement, CD28.ζ-CAR molecules rapidly but transiently translocated into mLRs, mobilizing the microtubular organizing center and lytic granules to the CARIS. This enabled fast CART recovery and sensitivity to low target site density. In contrast, gradual accumulation of 4-1BB.ζ-CAR and LFA-1 molecules at mLRs built mechanically tonic CARIS mediating chronic Fas ligand-based killing. The differences in CD28.ζ- and 4-1BB.ζ-CARIS dynamics explain the distinct cytolytic behavior of CART and can guide engineering of more adaptive effective cellular products.

We elucidate the mechanism underpinning a recently discovered phenomenon in which cells respond to MHz-order mechanostimuli. Deformations induced along the plasma membrane under these external mechanical cues are observed to decrease the membrane tension, which, in turn, drives transient and reversible remodeling of its lipid structure. In particular, the increase and consequent coalescence of ordered lipid microdomains leads to closer proximity to mechanosensitive ion channels-Piezo1, in particular-that, due to crowding, results in their activation to mobilize influx of calcium (Ca 2+ ) ions into the cell. It is the modulation of this second messenger that is responsible for the downstream signaling and cell fates that ensue. In addition, we show that such spatiotemporal control over the membrane microdomains in cells-without necessitating biochemical factors-facilitates aggregation and association of intrinsically disordered tau proteins in neuroblastoma cells, and their transformation to pathological conditions implicated in neurodegenerative diseases, thereby paving the way for the development of therapeutic intervention strategies., Competing Interests: Declaration of interests Authors declare that they have no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Introduction: Iron-mediated cell death (ferroptosis) is a proposed mechanism of Alzheimer's disease (AD) pathology. While iron is essential for basic biological functions, its reactivity generates oxidants which contribute to cell damage and death., Methods: To further resolve mechanisms of iron-mediated toxicity in AD, we analyzed post mortem human brain and ApoEFAD mice., Results: AD brains had decreased antioxidant enzymes, including those mediated by glutathione (GSH). Subcellular analyses of AD brains showed greater oxidative damage and lower antioxidant enzymes in lipid rafts, the site of amyloid processing, than in the non-raft membrane fraction. Apolipoprotein E ε4 carriers had lower lipid raft yield with greater membrane oxidation. The hypothesized role of iron in AD pathology was tested in ApoEFAD mice by iron chelation with deferoxamine, which decreased fibrillar amyloid and lipid peroxidation, together with increased GSH-mediated antioxidants., Discussion: These novel molecular pathways highlight iron-mediated damage to lipid rafts during AD., Highlghts: Alzheimer's disease (AD) brains have numerous markers for ferroptosis, including increased lipid peroxidation, reduced antioxidant levels, and increased iron storage. Lipid rafts in AD cases have increased oxidative damage and reduced antioxidant enzyme levels and activity which are most severe in apolipoprotein E ε4 carriers. Neuronal markers are correlated with lipid peroxidation, antioxidant defense, and iron signaling proteins suggesting that neuronal loss is linked to these events. Chelation of iron in the early-onset familial AD model reduces iron-mediated lipid peroxidation and fibrillar amyloid., (© 2025 The Author(s). Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Flotillin 1 and 2 are highly conserved and homologous members of the stomatin, prohibitin, flotillin, HflK/C (SPFH) family. These ubiquitous proteins assemble into hetero-oligomers at the cytoplasmic membrane in sphingolipid-enriched domains. Flotillins play crucial roles in various cellular processes, likely by concentrating sphingosine. They primarily act as scaffolding protein complexes within membrane microdomains (also called lipid rafts) and induce endocytosis and trafficking. Their diverse cargos in the upregulated flotillin-induced trafficking (UFIT) pathway, including tyrosine kinase receptors, adhesion molecules, and neurotransmitter receptors, link them to a wide range of cellular processes and diseases. Consequently, flotillin upregulation has been associated with various pathological conditions such as cancer, metabolic disorders, and neurodegenerative diseases. Flotillins may also be co-opted by pathogens to facilitate their entry and growth within host cells. In this review, we examined recent advancements in elucidating the structure and functions of the flotillin protein complex, including its implications in favoring the generation of sphingosine 1-phosphate, an essential bioactive lipid. We emphasized how the recent cryo-electron microscopy (cryo-EM) structure of a truncated cone-shaped cage composed of 22 copies of flotillin 1 and 2 subunits has enhanced our understanding of the flotillin complex organization within membrane microdomains and its role in membrane remodeling. We also explored how flotillin upregulation can perturb endosomal trafficking and contribute to various pathologies. A comprehensive understanding of flotillin oligomer organization and function is crucial to developing targeted therapies for diseases associated with flotillin overexpression., (© 2025 The Author(s). Biology of the Cell published by Wiley‐VCH GmbH on behalf of Société Française des Microscopies and Société de Biologie Cellulaire de France.)

NADPH oxidase 2 (Nox2), a superoxide-generating enzyme, is a source of reactive oxygen species (ROS) that regulate the intracellular redox state, self-renewal, and fate of hematopoietic stem/progenitor cells (HSPCs). Nox2 complex expressed on HSPCs associated with several activated cell membrane receptors increases the intracellular level of ROS. In addition, ROS are also released from mitochondria and, all together, are potent activators of intracellular pattern recognition receptor Nlrp3 inflammasome, which regulates the trafficking, proliferation, and metabolism of HSPCs. In the current study, we noticed that Nox2-deficient mice, despite the increased number of HSPCs in the bone marrow (BM), show hematopoietic defects illustrated by delayed recovery of peripheral blood (PB) hematopoietic parameters after sublethal irradiation and mobilize fewer HSPCs after administration of G-CSF and AMD3100. Moreover, Nox2-deficient HSPCs engraft poorly after transplantation into normal syngeneic recipients. To explain these defects at the molecular level, we hypothesized that Nox2-KO decreased ROS level does not efficiently activate Nlrp3 inflammasome, which plays a crucial role in regulating the trafficking of HSPCs. Herein, we report Nox2-deficient HSPCs display i) defective migration to major chemoattractant, ii) impaired intracellular activation of Nlrp3 inflammasome, and iii) a defect in membrane lipid raft (MLRs) formation that is required for a proper chemotactic response to pro-migratory factors. We conclude that Nox2-derived ROS enhances in Nlrp3 inflammasome-dependent manner HSPCs trafficking by facilitating MLRs assemble on the outer cell membranes, and defect in Nox2 expression results in impaired activation of Nlrp3 inflammasome, which affects HSPCs migration., Competing Interests: Declarations. Ethical Approval: Animal studies were approved by the Animal Care and Use Committee of the University of Louisville (Louisville, KY, USA) and the Warsaw Medical University (Warsaw, Poland). Competing Interests: None identified., (© 2024. The Author(s).)

The interactions between platelet-derived growth factor/PDGF receptor and integrin signaling are crucial for cells to respond to extracellular stimuli. Integrin interactions with PDGFR within the lipid rafts activate downstream cellular signaling pathways that regulate cell proliferation, cell migration, cell differentiation, and cell death processes. The mechanisms underlying PDGFR activation mediated receptor internalization, interactions with other cell-surface receptors, particularly extracellular matrix receptors, integrins, and how these cellular mechanisms switch on anchorage-independent cell survival, leading to anoikis resistance are discussed. The role of regulatory molecules such as noncoding RNAs, that can modulate several molecular and cellular processes, including autophagy, in the acquisition of anoikis resistance is also discussed. Overall, the review provides a new perspective on a complex interplay of regulatory cellular machineries involving autophagy, noncoding RNAs and cellular mechanisms of PDGFR activation, PDGFR-integrin interactions, and involvement of lipids rafts in the acquisition of anoikis resistance that regulates glioblastoma progression along with potential future strategies to develop novel therapeutics for glioblastoma multiforme., (© 2024 International Federation for Cell Biology.)

Cholesterol dysregulation, disorder of neuronal membrane lipid packing, and lipid rafts lead to the synthesis and accumulation of toxic amyloid-β (Aβ), contributing to the development of Alzheimer's disease (AD). Our study shows that near-infrared (NIR) transcranial photobiomodulation therapy (tPBMT) can reduce Aβ load and restore the properties of neuronal plasma membrane, including Aβ production, bilayer order, rafts, lipid content, and Ca 2+ channels during AD. Mice in the experiments were exposed to 808-nm LED for 1 h daily over 3 months. In the APOE transgenic model with cholesterol dysregulation, the cholesterol levels increased by 22 times, causing healthy neurons to produce toxic Aβ three times faster, increasing its load by five times. Consequently, Aβ disrupts the membrane bilayer and prompts the formation of lipid rafts and pores. NIR-tPBMT can nearly half attenuate Aβ load, restore membrane lipid order and rigidity, reduce the number of lipid rafts, modulate cholesterol synthesis, normalize Ca 2+ influx by activated endocytosis, and reduce neuronal death. The Ca 2+ influx induced by light does not cause excitotoxicity but modulates Ca 2+ /calmodulin signaling involved in AD mechanisms and cell viability. The transcriptome analysis of the brain cortex and hippocampus shows that light can downregulate Ca 2+ /calmodulin-dependent AD-risk genes BACE, PSEN, and APP, and normalize cholesterol homeostasis via the HMGCR, DHCR7, and INSIG1 genes. Additionally, light enhances neuron resistance to the endoplasmic reticulum stress via activating transcription factors of the unfolded protein response. Thus, red/NIR light could be promising in combating AD, restoring synaptic plasticity in degenerating neurons and reducing Aβ load., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lipid rafts are subdomains of the cell membrane that are rich in cholesterol and glycolipids, and they are involved in various cellular processes and pathophysiological mechanisms. However, the specific role of lipid rafts in hepatocyte dysfunction during the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD) is not fully understood. In this study, we investigated the impact of lipid rafts on insulin sensitivity and hepatocyte injury induced by saturated free fatty acids (sFFAs) using primary-cultured mouse hepatocytes. Treatment of primary hepatocytes with palmitic acid (PA) resulted in significant lipid droplet accumulation in the cytoplasm and a marked increase in the number of cells with accumulation of lipid raft. The addition of cholesterol oxidase (CHOX), which disrupts lipid rafts, did not affect PA-induced lipid droplet formation, but significantly reduced the number of cells with accumulation of lipid raft. In PA-treated hepatocytes, insulin-stimulated phosphorylation of insulin receptor substrate (IRS)-2 and Akt was markedly decreased, but this effect was alleviated by CHOX. Furthermore, PA-pretreated hepatocytes exhibited substantial increases in reactive oxygen species (ROS) production and cell death when exposed to low doses of tert-butyl hydroperoxide (t-BuOOH). In contrast, treatment with CHOX after PA significantly reduced ROS production and cell death following t-BuOOH. These results suggest that PA-induced accumulation of lipid raft not only exacerbates insulin resistance but also enhances responsiveness to oxidative stress stimuli, contributing to MASLD pathogenesis. Modulation of lipid rafts may represent a promising therapeutic target for MASLD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Toll-like receptor 4 (TLR4) and the transient receptor potential vanilloid subtype 1 (TRPV1) are both upregulated and play key roles in the induction and expression of paclitaxel-related chemotherapy-induced peripheral neuropathy (CIPN). Using Apolipoprotein A-I binding protein, non-specific cholesterol depletion, TLR4 mis-sense rats and a TLR4 inhibitor, we demonstrate that co-localization of TRPV1 with TLR4 to cholesterol-rich lipid membrane rafts in nociceptors is essential for its normal activation as well as for its exaggerated activation that underlies the development and expression of CIPN. The findings suggest that TLR4-lipid rafts may have an essential role in numerous neuroinflammatory and neuropathic pain conditions. This mechanism is also generalized to female rats for the first time., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The effects of a peripheral protein - cholesterol oxidase (3β-hydroxysteroid oxidase, ChOx) on the characteristics of model lipid membranes composed of cholesterol, cholesterol:sphingomyelin (1:1), and the raft model composed of DOPC:Chol:SM (1:1:1) were investigated using two membrane model systems: the flat monolayer prepared by the Langmuir technique and the curved model consisting of liposome of the same lipids. The planar monolayers and liposomes were employed to follow membrane cholesterol oxidation to cholestenone catalyzed by ChOx and changes in the lipid membrane structure accompanying this reaction. Changes in the structure of liposomes in the presence of the enzyme were reflected in the changes of hydrodynamic diameter and fluorescence microscopy images, while changes of surface properties of planar membranes were evaluated by grazing incidence X-ray diffraction (GIXD) and Brewster angle microscopy. UV-Vis absorbance measurements confirmed the activity of the enzyme in the tested systems. A better understanding of the interactions between the enzyme and the cell membrane may help in finding alternative ways to decrease excessive cholesterol levels than the common approach of treating hypercholesterolemia with statins, which are not free from undesirable side effects, repeatedly reported in the literature and observed by the patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Many membrane proteins are modulated by cholesterol. Here we report profound effects of cholesterol depletion and restoration on the human voltage-gated proton channel, hH V 1, in excised patches but negligible effects in the whole-cell configuration. Despite the presence of a putative cholesterol-binding site, a CARC motif in hH V 1, mutation of this motif did not affect cholesterol effects. The murine H V 1 lacks a CARC sequence but displays similar cholesterol effects. These results argue against a direct effect of cholesterol on the H V 1 protein. However, the data are fully explainable if H V 1 preferentially associates with cholesterol-dependent lipid domains, or "rafts." The rafts would be expected to concentrate in the membrane/glass interface and to be depleted from the electrically accessible patch membrane. This idea is supported by evidence that H V 1 channels can diffuse between seal and patch membranes when suction is applied. Simultaneous truncation of the large intracellular N and C termini of hH V 1 greatly attenuated the cholesterol effect, but C truncation alone did not; this suggests that the N terminus is the region of attachment to lipid domains. Searching for abundant raft-associated proteins led to stomatin. Co-immunoprecipitation experiment results were consistent with hH V 1 binding to stomatin. The stomatin-mediated association of H V 1 with cholesterol-dependent lipid domains provides a mechanism for cells to direct H V 1 to subcellular locations where it is needed, such as the phagosome in leukocytes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Regarding flotillin knockdown, drug resistance is reversed in colorectal cancer (CRC) cell lines; this is associated with the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway, as our previous experimental results indicated. However, the exact mechanism underlying this pathway remains unclear. PI3K inhibitor and activator were added separately to clarify the role of the PI3K pathway in reversing drug resistance. The results showed decreased resistance after inhibiting PI3K activity. Furthermore, the reduced resistance due to flotillin knockdown was restored after adding the PI3K activator. Additional results showed no changes in PI3K molecules. However, p-AKT expression was downregulated. Further results suggested that the phosphatidylinositol (3,4,5)-trisphosphate/phosphatidylinositol 4,5-bisphosphate (PIP3/PIP2) ratio was downregulated, whereas the phosphatase and tensin homolog (PTEN) expression was upregulated. In addition, we also found that P-gp activity inhibition resulted in increased adriamycin accumulation and reversal of resistance, and flotillin knockdown was accompanied by a downregulation of P-gp expression in CRC cells. In conclusion, our study demonstrated that flotillin knockdown could reverse drug resistance in CRC cells by downregulating the PTEN/PI3K/AKT pathway and P-gp.

Cholesterol- and sphingolipid-enriched domains called lipid rafts are hypothesized to selectively coordinate protein complex assembly within the plasma membrane to regulate cellular functions. Desmosomes are mechanically resilient adhesive junctions that associate with lipid raft membrane domains, yet the mechanisms directing raft association of the desmosomal proteins, particularly the transmembrane desmosomal cadherins, are poorly understood. We identified the desmoglein-1 (DSG1) transmembrane domain (TMD) as a key determinant of desmoglein lipid raft association and designed a panel of DSG1 TMD variants to assess the contribution of TMD physicochemical properties (length, bulkiness, and palmitoylation) to DSG1 lipid raft association. Sucrose gradient fractionations revealed that TMD length and bulkiness, but not palmitoylation, govern DSG1 lipid raft association. Further, DSG1 raft association determines plakoglobin recruitment to raft domains. Super-resolution imaging and functional assays uncovered a strong relationship between the efficiency of DSG1 TMD lipid raft association and the formation of morphologically and functionally robust desmosomes. Lipid raft association regulated both desmosome assembly dynamics and DSG1 cell surface stability, indicating that DSG1 lipid raft association is required for both desmosome formation and maintenance. These studies identify the biophysical properties of desmoglein transmembrane domains as key determinants of lipid raft association and desmosome adhesive function., Competing Interests: Conflicts of interests: The authors declare no financial conflict of interest.

Ca 2+ signalling is pivotal in T cell activation, an essential process in adaptive immune responses. Key to this activation are Ca 2+ microdomains, which are transient increases in cytosolic Ca 2+ concentration occurring within narrow regions between the endoplasmic reticulum (ER) and the plasma membrane (PM), lasting a few tens of milliseconds. Adhesion Dependent Ca 2+ Microdomains (ADCM) rely on store-operated Ca 2+ entry (SOCE) via the ORAI/STIM system. The nanometric scale at which these microdomains form poses challenges for direct experimental observation. Following the previous work of Gil et al. [1], which introduced a three-dimensional model of the ER-PM junction, this study combines a detailed description of the Ca 2+ fluxes at the junction with stochastic dynamics of a cluster of D-myo-inositol 1,4,5 trisphosphate receptors (IP 3 R) located in the ER surrounding the junction. Because the consideration of Ca 2+ release through the IP 3 R calls for the simulation of a portion of the cytoplasm considerably larger than the junction, our study also investigates the spatial distribution of PMCAs, revealing their likely localization outside the ER-PM junction. Simulations indicate that Ca 2+ puffs implying the opening of 2-6 IP 3 Rs create ADCMs by provoking local depletions of ER Ca 2+ stimulating Ca 2+ entry through the ORAI1 channels. Such conditions allow the reproduction of the amplitude, duration and spatial extent of the observed ADCMs. By integrating advanced computational techniques with insights from experimental studies, our approach provides valuable information on the mechanisms governing early Ca 2+ signalling in T cell activation, paving the way for a deeper understanding of immune responses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The proinflammatory cytokine interferon gamma (IFNγ) is upregulated in a variety of infections and contributes to bone marrow failure through hematopoietic stem cell (HSC) activation and subsequent exhaustion. The cell-surface protein, bone marrow stromal antigen 2 (BST2), is a key mediator of this process, because it is induced upon IFN stimulation and required for IFN-dependent HSC activation. To identify the mechanism by which BST2 promotes IFN-dependent HSC activation, we evaluated its role in niche localization, immune cell function, lipid raft formation, and intracellular signaling. Our studies indicated that knockout (KO) of BST2 in a murine model does not disrupt immune cell responses to IFN-inducing mycobacterial infection. Furthermore, intravital imaging studies indicate that BST2 KO does not disrupt localization of HSCs relative to endothelial or osteoblastic niches in the bone marrow. However, using imaging-based flow cytometry, we found that IFNγ treatment shifts the lipid raft polarity of wild-type (WT) but not Bst2 -/- hematopoietic stem and progenitor cells (HSPCs). Furthermore, RNAseq analysis, reverse-phase protein array and western blot analysis of HSPCs indicate that BST2 promotes ERK1/2 phosphorylation during IFNγ-mediated stress. Overall, we find that BST2 facilitates HSC division by promoting cell polarization and ERK activation, thus elucidating a key mechanism of IFN-dependent HSPC activation. These findings inform future approaches in the treatment of cancer and bone marrow failure., Competing Interests: Conflicts of Interest Disclosure The authors declare no competing interests., (Copyright © 2024 International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Most studies on the docking of ivermectin on the spike protein of SARS-CoV-2 concern the receptor binding domain (RBD) and, more precisely, the RBD interface recognized by the ACE2 receptor. The N-terminal domain (NTD), which controls the initial attachment of the virus to lipid raft gangliosides, has not received the attention it deserves. In this study, we combined molecular modeling and physicochemical approaches to analyze the mode of interaction of ivermectin with the interface of the NTD-facing lipid rafts of the host cell membrane. We characterize a binding area that presents point mutations and deletions in successive SARS-CoV-2 variants from the initial strain to omicron KP.3 circulating in many countries in 2024. We show that ivermectin has exceptional flexibility, allowing the drug to bind to the spike protein of all variants tested. The energy of interaction is specific to each variant, allowing a classification according to their affinity for ivermectin in the following ascending order: Omicron KP.3 < Delta < Omicron BA.5 < Alpha < Wuhan (B.1) < Omicron BA.1. The binding site of ivermectin is subject to important variations of the NTD, including the Y144 deletion. It overlaps with the ganglioside binding domain of the NTD, as demonstrated by docking and physicochemical studies. These results suggest a new mechanism of antiviral action for ivermectin based on competitive inhibition for initial virus attachment to lipid rafts. The current KP.3 variant is still recognized by ivermectin, although with an affinity slightly lower than the Wuhan strain.

Cell membranes consist of heterogeneous lipid nanodomains that influence key cellular processes. Using FRET-based fluorescent assays and fluorescence lifetime imaging microscopy (FLIM), we find that the dimension of cholesterol-enriched ordered membrane domains (OMD) varies considerably, depending on specific cell types. Particularly, nociceptor dorsal root ganglion (DRG) neurons exhibit large OMDs. Disruption of OMDs potentiated action potential firing in nociceptor DRG neurons and facilitated the opening of native hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels. This increased neuronal firing is partially due to an increased open probability and altered gating kinetics of HCN channels. The gating effect on HCN channels is likely due to a direct modulation of their voltage sensors by OMDs. In animal models of neuropathic pain, we observe reduced OMD size and a loss of HCN channel localization within OMDs. Additionally, cholesterol supplementation inhibited HCN channels and reduced neuronal hyperexcitability in pain models. These findings suggest that disturbances in lipid nanodomains play a critical role in regulating HCN channels within nociceptor DRG neurons, influencing pain modulation., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The mammalian plasma membrane is known to contain domains with varying lipid composition and biophysical properties. However, studying these membrane lipid domains presents challenges due to their predicted morphological similarity to the bulk membrane and their scale being below the classical resolution limit of optical microscopy. To address this, we combine the solvatochromic probe di-4-ANEPPDHQ, which reports on its biophysical environment through changes in its fluorescence emission, with spectrally resolved single-molecule localisation microscopy. The resulting data comprises nanometre-precision localisation coordinates and a generalised polarisation value related to the probe's environment - a marked point pattern. We introduce quantification algorithms based on topological data analysis (PLASMA) to detect and map nano-domains in this marked data, demonstrating their effectiveness in both artificial membranes and live cells. By leveraging environmentally sensitive fluorophores, multi-modal single molecule localisation microscopy, and advanced analysis methods, we achieve nanometre scale mapping of membrane properties and assess changes in response to external perturbation with methyl-β-cyclodextrin. This integrated methodology represents an integrated toolset for investigating marked point pattern data at nanometre spatial scales., (© 2024. The Author(s).)

The endoplasmic reticulum is organized into ordered regions enriched in cholesterol and sphingomyelin, and disordered microdomains characterized by more fluidity. Rabbit CYP1A1 and CYP1A2 localize into disordered and ordered microdomains, respectively. Previously, a CYP1A2 chimera containing the first 109 amino acids of CYP1A1 showed altered microdomain localization. The goal of this study was to identify specific residues responsible for CYP1A microdomain localization. Thus, CYP1A2 chimeras containing substitutions from homologous regions of CYP1A1 were expressed in HEK 293T/17 cells, and the localization was examined after solubilization with Brij 98. A CYP1A2 mutant with the three amino acids from CYP1A1 (VAG) at positions 27 to 29 of CYP1A2 was generated that showed a distribution pattern similar to those of CYP1A1/1A2 chimeras containing both the first 109 amino acids and the first 31 amino acids of CYP1A1 followed by remaining amino acids of CYP1A2. Similarly, the reciprocal substitution of three amino acids from CYP1A2 (AVR) into CYP1A1 resulted in a partial redistribution of the chimera into ordered microdomains. Molecular dynamic simulations indicate that the positive charges of the CYP1A1 and CYP1A2 linker regions between the N termini and catalytic domains resulted in different depths of immersion of the N termini in the membrane. The overlap of the distribution of positively charged residues in CYP1A2 (AVR) and negatively charged phospholipids was higher in the ordered than in the disordered microdomain. These findings identify three residues in the CYP1AN terminus as a novel microdomain-targeting motif of the P450s and provide a mechanistic explanation for the differential microdomain localization of CYP1A., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Severe fever with thrombocytopenia syndrome virus (SFTSV), recently named as Dabie bandavirus, belongs to the family Phenuiviridae of the order Bunyavirales, is a newly-identified bunyavirus with a case fatality rate of up to 30%, posing a serious threat to public health. Lipid rafts on plasm membranes are important for the entry of enveloped viruses; however, the role of lipid rafts in bunyavirus entry remains unclear. In this study, we found that methyl-beta-cyclodextrin (MβCD), a drug that disrupts cholesterol in lipid rafts of cell membranes, inhibits SFTSV infection. Additionally, there is a back-complementary effect of SFTSV infection upon the addition of cholesterol. Moreover, the concentration of SFTSV particles in lipid rafts during entry directly indicated the role of lipid rafts as a gateway, whereas MβCD could inhibit SFTSV entry by affecting the structure of lipid rafts. In an in vivo study, MβCD also reduced the susceptibility of mice to SFTSV infection. Our results suggest that SFTSV can interact with Talin1 proteins on lipid rafts to enter host cells by endocytosis of lipid rafts and reveal the potential therapeutic value of MβCD for SFTSV infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

During cell invasion, large Extracellular Vesicle (lEV) release from host cells was dose-dependently triggered by Trypanosoma cruzi metacyclic trypomastigotes (Mtr). This lEV release was inhibited when IP 3 -mediated Ca 2+ exit from the ER and further Ca 2+ entry from plasma membrane channels was blocked, but whilst any store-independent Ca 2+ entry (SICE) could continue unabated. That lEV release was equally inhibited if all entry from external sources was blocked by chelation of external Ca 2+ points to the major contributor to Mtr-triggered host cell lEV release being IP 3 /store-mediated Ca 2+ release, SICE playing a minor role. Host cell lEVs were released through Mtr interaction with host cell lipid raft domains, integrins, and mechanosensitive ion channels, whereupon [Ca 2+ ] cyt increased (50 to 750 nM) within 15 s. lEV release and cell entry of T. cruzi, which increased up to 30 and 60 mpi, respectively, as well as raised actin depolymerization at 60 mpi, were all reduced by TRPC inhibitor, GsMTx-4. Vesicle release and infection was also reduced with RGD peptide, methyl-β-cyclodextrin, knockdown of calpain and with the calpain inhibitor, calpeptin. Restoration of lEV levels, whether with lEVs from infected or uninfected epithelial cells, did not restore invasion, but supplementation with lEVs from infected monocytes, did. We provide evidence of THP-1 monocyte-derived lEV interaction with Mtr (lipid mixing by R18-dequenching; flow cytometry showing transfer to Mtr of R18 from R18-lEVs and of LAP(TGF-β1). Active, mature TGF-β1 (at 175 pg/×10 5 in THP-1 lEVs) was detected in concentrated lEV-/cell-free supernatant by western blotting, only after THP-1 lEVs had interacted with Mtr. The TGF-β1 receptor (TβRI) inhibitor, SB-431542, reduced the enhanced cellular invasion due to monocyte-lEVs., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Aging induces complex changes in the lipid profiles across different areas of the brain. These changes can affect the function of brain cells and may contribute to neurodegenerative diseases such as Alzheimer's disease. Research shows that while the overall lipid profile in the human brain remains quite steady throughout adulthood, specific changes occur with age, especially after the age of 50. These changes include a slow decline in total lipid content and shifts in the composition of fatty acids, particularly in glycerophospholipids and cholesterol levels, which can vary depending on the brain region. Lipid rafts play a crucial role in maintaining membrane integrity and facilitating cellular signaling. In the context of Alzheimer's disease, changes in the composition of lipid rafts have been associated with the development of the disease. For example, alterations in lipid raft composition can lead to increased accumulation of amyloid β (Aβ) peptides, contributing to neurotoxic effects. Lipid droplets store neutral lipids and are key for cellular energy metabolism. As organisms age, the dynamics of lipid droplets in the brain change, with evidence suggesting a decline in metabolic activity over time. This reduced activity may lead to an imbalance in lipid synthesis and mobilization, contributing to neurodegenerative processes. In model organisms like Drosophila, studies have shown that lipid metabolism in the brain can be influenced by diet and insulin signaling pathways, crucial for maintaining metabolic balance. The interplay between lipid metabolism, oxidative stress, and inflammation is critical in the context of aging and Alzheimer's disease. Lipid peroxidation, a consequence of oxidative stress, can lead to the formation of reactive aldehydes that further damage neurons. Inflammatory processes can also disrupt lipid metabolism, contributing to the pathology of AD. Consequently, the accumulation of oxidized lipids can affect lipid raft integrity, influencing signaling pathways involved in neuronal survival and function.

Abstract: Patients with thrombocytopenia require platelet transfusion to prevent and stop hemorrhage. Cold storage of platelets results in complex molecular lesions, including changes in membrane microdomains that are recognized by host macrophages and hepatocyte counter-receptors, resulting in phagocytosis and clearance upon transfusion. For this reason, platelets are stored at room temperature, a method that confers increased risk of bacterial contamination. By applying signaling analysis and genetic and pharmacological approaches, we identified that cold-induced activation of RAS homolog family, member A (RHOA) GTPase causes the major hallmarks of platelet cold storage lesions. RHOA deficiency renders murine platelets insensitive to cold storage-induced damage, and pharmacological inhibition by a RHOA activation inhibitor, R-G04, can prevent the cold storage-induced lesions. RHOA inhibition prevents myosin activation and clathrin-independent formation and internalization of lipid rafts enriched in active glycosyltransferases as well as abnormal distribution of GPIbα. RHOA inhibition further prevents the metabolic reprogramming of cold storage-induced lesions and allows the maintenance of glycolytic flux and mitochondria-dependent respiration. Importantly, human platelets transfused in mice after cold storage, in the presence of R-G04 or its more potent enantiomer S-G04, can circulate in vivo at similar levels as room temperature-stored platelets while retaining their hemostatic activity in vivo, as assessed by bleeding time correction in aspirin-treated mice. Our studies provide a mechanism-based translational approach to prevent cold storage-induced damage, which is useful for human platelet transfusion in patients with thrombocytopenia., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Staphylococcus aureus is a pathogen associated with severe respiratory infections. The ability of S. aureus to internalize into lung epithelial cells complicates the treatment of respiratory infections caused by this bacterium. In the intracellular environment, S. aureus can avoid elimination by the immune system and the action of circulating antibiotics. Consequently, interfering with S. aureus internalization may represent a promising adjunctive therapeutic strategy to enhance the efficacy of conventional treatments. Here, we investigated the host-pathogen molecular interactions involved in S. aureus internalization into human lung epithelial cells. Lipid raft-mediated endocytosis was identified as the main entry mechanism. Thus, bacterial internalization was significantly reduced after the disruption of lipid rafts with methyl-β-cyclodextrin. Confocal microscopy confirmed the colocalization of S. aureus with lipid raft markers such as ganglioside GM1 and caveolin-1. Adhesion of S. aureus to α5β1 integrin on lung epithelial cells via fibronectin-binding proteins (FnBPs) was a prerequisite for bacterial internalization. A mutant S. aureus strain deficient in the expression of alpha-hemolysin (Hla) was significantly impaired in its capacity to enter lung epithelial cells despite retaining its capacity to adhere. This suggests a direct involvement of Hla in the bacterial internalization process. Among the receptors for Hla located in lipid rafts, caveolin-1 was essential for S. aureus internalization, whereas ADAM10 was dispensable for this process. In conclusion, this study supports a significant role of lipid rafts in S. aureus internalization into human lung epithelial cells and highlights the interaction between bacterial Hla and host caveolin-1 as crucial for the internalization process., (© 2024. The Author(s).)

Lipid rafts are dynamic microdomains enriched with cholesterol and sphingolipids that play critical roles in cellular processes by organizing and concentrating specific proteins involved in signal transduction. The interplay between lipid rafts, raft-associated caveolae and the human epidermal growth factor receptors has significant implications in cancer biology, particularly in breast and gastric cancer therapy resistance. This review examines the structural and functional characteristics of lipid rafts, their involvement in EGFR and HER2 signaling, and the impact of lipid rafts/CXCL12/CXCR4/HER2 axis on bone metastasis. We also discuss the potential of targeting lipid rafts and caveolin-1 to enhance therapeutic strategies against HER2-positive cancers and the impact of co-localization of trastuzumab or antibody drug conjugates with caveolin-1 on therapy response. Emerging evidence suggests that disrupting lipid raft integrity or silencing caveolin-1, through several strategies including cholesterol-lowering molecules, can influence HER2 availability and internalization, enhancing anti-HER2 targeted therapy and offering a novel approach to counteract drug resistance and improve treatment efficacy., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

Tetraspanins (TSPANs) are a family of highly conserved proteins present in a wide variety of eukaryotes. Although protein-protein interactions of TSPANs have been well established in eukaryotes including parasitic protists, the role they play in parasitism and pathogenesis remains largely unknown. In this study, we characterized three representative members of TSPANs, TSPAN4, TSPAN12, and TSPAN13 from the human intestinal protozoan Entamoeba histolytica. Co-immunoprecipitation assays demonstrated that TSPAN4, TSPAN12 and TSPAN13 are reciprocally pulled down together with several other TSPAN-interacting proteins including TSPAN binding protein of 55kDa (TBP55) and interaptin. Blue native-PAGE analysis showed that these TSPANs form several complexes of 120-250 kDa. Repression of tspan12 and tspan13 gene expression led to decreased secretion of cysteine proteases, while repression of tspan4 led to a four-fold increase in the activity of cysteine proteases in crude extracellular vesicles (EVs) fraction. Meanwhile, strains overexpressing HA-tagged TSPAN12 and TSPAN13 demonstrated reduced adhesion to collagen. Altogether, this study reveals that the TSPANs, especially TSPAN12 and TSPAN13, are engaged with complex protein-protein interactions and are involved in the pathogenicity-related biological functions such as protease secretion and adhesion, offering insights into the potential regulatory mechanisms of tetraspanins in protozoan parasites., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Jiang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2024. The Author(s).)

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 Cys 17,18 and Cys 246 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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

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., Competing Interests: PC, JO, Bd, CA, TW No competing interests declared, (© 2023, Chiu, Orjuela et al.)

Euphorbia lathyris L. (EL) is a traditional poisonous herbal medicine used to treat dropsy, ascites, amenorrhea, anuria and constipation. Processing to reduce toxicity of EL is essential for its safe and effective application. However, there is little known regarding the molecular mechanism of reducing toxicity after EL processing. This research aimed to screen the differential markers for EL and PEL, explore the differential mechanisms of inflammatory injury induced by EL and processed EL (PEL) to expound the mechanism of alleviating toxicity after EL processing. The results showed that 15 potential biomarkers, mainly belonging to diterpenoids, were screened to distinguish EL from PEL. EL promoted the expressions of TLR4, NLRP3, NF-κB p65, IL-1β and TNF-α, increased lipid rafts abundance and promoted TLR4 positioning to lipid rafts. Meanwhile, EL decreased LXRα and ABCA1 expression, and reduced cholesterol efflux. In contrast to EL, the effects of PEL on these indicators were markedly weakened. In addition, Euphorbia factors L 1 , L 2 , and L 3 affected LXRα, ABCA1, TLR4, NLRP3, NF-κB p65, TNF-α and IL-1β expression, influenced cholesterol efflux and lipid rafts abundance, and interfered with the colocalization of TLR4 and lipid rafts. The inflammatory injury caused by processed EL was significantly weaker than that caused by crude EL, and reduction of Euphorbia factors L 1 , L 2 , and L 3 as well as attenuation of inflammatory injury participated in processing-based detoxification of EL. Our results provide valuable insights into the attenuated mechanism of EL processing and will guide future research on the processing mechanism of toxic traditional Chinese medicine., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest regarding the publication of this paper., (Copyright © 2023. Published by Elsevier B.V.)

Aims: Despite aggressive treatment, the recurrence of glioma is an inevitable occurrence, leading to unsatisfactory clinical outcomes. A plausible explanation for this phenomenon is the phenotypic alterations that glioma cells undergo aggressive therapies, such as TMZ-therapy. However, the underlying mechanisms behind these changes are not well understood., Methods: The TMZ chemotherapy resistance model was employed to assess the expression of intercellular adhesion molecule-1 (ICAM1) in both in vitro and in vivo settings. The potential role of ICAM1 in regulating TMZ chemotherapy resistance was investigated through knockout and overexpression techniques. Furthermore, the mechanism underlying ICAM1-mediated TMZ chemotherapy resistance was examined using diverse molecular biological methods, and the lipid raft protein was subsequently isolated to investigate the cellular subcomponents where ICAM1 operates., Results: Acquired TMZ resistant (TMZ-R) glioma models heightened production of intercellular adhesion molecule-1 (ICAM1) in TMZ-R glioma cells. Additionally, we observed a significant suppression of TMZ-R glioma proliferation upon inhibition of ICAM1, which was attributed to the enhanced intracellular accumulation of TMZ. Our findings provide evidence supporting the role of ICAM1, a proinflammatory marker, in promoting the expression of ABCB1 on the cell membrane of TMZ-resistant cells. We have elucidated the mechanistic pathway by which ICAM1 modulates phosphorylated moesin, leading to an increase in ABCB1 expression on the membrane. Furthermore, our research has revealed that the regulation of moesin by ICAM1 was instrumental in facilitating the assembly of ABCB1 exclusively on the lipid raft of the membrane., Conclusions: Our findings suggest that ICAM1 is an important mediator in TMZ-resistant gliomas and targeting ICAM1 may provide a new strategy for enhancing the efficacy of TMZ therapy against glioma., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rong Hu reports administrative support, article publishing charges, equipment, drugs, or supplies, statistical analysis, and writing assistance were provided by National Natural Science Foundation of China. Qing Wang reports administrative support, article publishing charges, equipment, drugs, or supplies, statistical analysis, travel, and writing assistance were provided by Natural Science Foundation of Jiangsu Province., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

β2-Adrenoceptors (β2-ARs) are the most abundant subtype of adrenergic receptors in skeletal muscles. Their activation via a stabilization of postsynaptic architecture has beneficial effects in certain models of neuromuscular disorders. However, the ability of β2-ARs to regulate neuromuscular transmission at the presynaptic level is poorly understood. Using electrophysiological recordings and fluorescent FM dyes, we found that β2-AR activation with fenoterol enhanced an involvement of synaptic vesicles in exocytosis and neurotransmitter release during intense activity at the neuromuscular junctions of mouse diaphragm. This was accompanied by an improvement of contractile responses to phrenic nerve stimulation (but not direct stimulation of the muscle fibers) at moderate-to-high frequencies. β2-ARs mainly reside in lipid microdomains enriched with cholesterol and sphingomyelin. The latter is hydrolyzed by sphingomyelinases, whose upregulation occurs in many conditions characterized by muscle atrophy and sympathetic nerve hyperactivity. Sphingomyelinase treatment reversed the effects of β2-AR agonist on the neurotransmitter release and synaptic vesicle recruitment to the exocytosis during intense activity. Inhibition of G i protein with pertussis toxin completely prevented the sphingomyelinase-mediated inversion in the β2-AR agonist action. Note that lipid raft disrupting enzyme cholesterol oxidase had the same effect on β2-AR agonist-mediated changes in neurotransmission as sphingomyelinase. Thus, β2-AR agonist fenoterol augmented recruitment and release of synaptic vesicles during intense activity in the diaphragm neuromuscular junctions. Sphingomyelin hydrolysis inversed the effects of β2-AR agonist on neurotransmission probably via switching to G i protein-dependent signaling. This phenomenon may reflect a dependence of the β2-AR signaling on lipid raft integrity in the neuromuscular junctions., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The neuronal glycine transporter GlyT2 removes glycine from the synaptic cleft through active Na + , Cl - , and glycine cotransport contributing to the termination of the glycinergic signal as well as supplying substrate to the presynaptic terminal for the maintenance of the neurotransmitter content in synaptic vesicles. Patients with mutations in the human GlyT2 gene (SLC6A5), develop hyperekplexia or startle disease (OMIM 149400), characterized by hypertonia and exaggerated startle responses to trivial stimuli that may have lethal consequences in the neonates as a result of apnea episodes. Post-translational modifications in cysteine residues of GlyT2 are an aspect of structural interest we analyzed. Our study is compatible with a reversible and short-lived S-acylation in spinal cord membranes, detectable by biochemical and proteomics methods (acyl-Rac binding and IP-ABE) confirmed with positive and negative controls (palmitoylated and non-palmitoylated proteins). According to a short-lived modification, direct labeling using click chemistry was faint but mostly consistent. We have analyzed the physiological properties of a GlyT2 mutant lacking the cysteines with high prediction of palmitoylation and the mutant is less prone to be included in lipid rafts, an effect also observed upon treatment with the palmitoylation inhibitor 2-bromopalmitate. This work demonstrates there are determinants of lipid raft inclusion associated with the GlyT2 mutated cysteines, which are presumably modified by palmitoylation., (© 2024 The Author(s). Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

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., (Copyright © 2024 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

Like 'influencers' who achieve fame and power through social media, ceramides are low abundance members of communication platforms that have a mighty impact on their surroundings. Ceramide microdomains form within sphingolipid-laden lipid rafts that confer detergent resistance to cell membranes and serve as important signaling hubs. In cells exposed to excessive amounts of saturated fatty acids (e.g. in obesity), the abundance of ceramide-rich microdomains within these rafts increases, leading to concomitant alterations in cellular metabolism and survival that contribute to cardiometabolic disease. In this mini-review, we discuss the evidence supporting the formation of these ceramide microdomains and describe the spectrum of harmful ceramide-driven metabolic actions under the context of an evolutionary theory. Moreover, we discuss the proximal 'followers' of these ceramide media stars that account for the diverse intracellular actions that allow them to influence obesity-linked disease., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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 ( 1 O 2 ) 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.

Annexin A2 (A2)-induced microdomain formation is a key step in biological processes such as Ca 2+ -mediated exocytosis in neuroendocrine cells. In this work, a total of 15 coarse-grained molecular dynamics simulations were performed on vesicle models having a diameter of approximately 250 Å for 15 μs each using the Martini2 force field. Five simulations were performed in the presence of 10 A2, 5 in the presence of A2 but absence of PIP 2 , and 5 simulations in the absence of A2 but presence of PIP 2 . Consistent results were generated among the simulations. A2-induced PIP 2 microdomain formation was observed and shown to occur in three phases: A2-vesicle association, localized A2-induced PIP 2 clustering, and A2 aggregation driving PIP 2 microdomain formation. The relationship between A2 aggregation and PIP 2 microdomain formation was quantitatively described using a novel method which calculated the variance among protein and lipid positions via the Fréchet mean. A large reduction in PIP 2 variance was observed in the presence of A2 but not in its absence. This reduction in PIP 2 variance was proportional to the reduction observed in A2 variance and demonstrates that the observed PIP 2 microdomain formation is dependent upon A2 aggregation. The three-phase model of A2-induced microdomain formation generated in this work will serve as a valuable guide for further experimental studies and the development of novel A2 inhibitors. No microdomain formation was observed in the absence of A2 and minimal A2-membrane interaction was observed in the absence of PIP 2 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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., (© 2024. Springer Nature Limited.)

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., (©2024 The Authors; Published by the American Association for Cancer Research.)

Acetylcholine is the main neurotransmitter at the vertebrate neuromuscular junctions (NMJs). ACh exocytosis is precisely modulated by co-transmitter ATP and its metabolites. It is assumed that ATP/ADP effects on ACh release rely on activation of presynaptic G i protein-coupled P 2 Y 13 receptors. However, downstream signaling mechanism of ATP/ADP-mediated modulation of neuromuscular transmission remains elusive. Using microelectrode recording and fluorescent indicators, the mechanism underlying purinergic regulation was studied in the mouse diaphragm NMJs. Pharmacological stimulation of purinoceptors with ADP decreased synaptic vesicle exocytosis evoked by both low and higher frequency stimulation. This inhibitory action was suppressed by antagonists of P 2 Y 13 receptors (MRS 2211), Ca 2+ mobilization (TMB8), protein kinase C (chelerythrine) and NADPH oxidase (VAS2870) as well as antioxidants. This suggests the participation of Ca 2+ and reactive oxygen species (ROS) in the ADP-triggered signaling. Indeed, ADP caused an increase in cytosolic Ca 2+ with subsequent elevation of ROS levels. The elevation of [Ca 2+ ] in was blocked by MRS 2211 and TMB8, whereas upregulation of ROS was prevented by pertussis toxin (inhibitor of G i protein) and VAS2870. Targeting the main components of lipid rafts, cholesterol and sphingomyelin, suppressed P 2 Y 13 receptor-dependent attenuation of exocytosis and ADP-induced enhancement of ROS production. Inhibition of P 2 Y 13 receptors decreased ROS production and increased the rate of exocytosis during intense activity. Thus, suppression of neuromuscular transmission by exogenous ADP or endogenous ATP can rely on P 2 Y 13 receptor/G i protein/Ca 2+ /protein kinase C/NADPH oxidase/ROS signaling, which is coordinated in a lipid raft-dependent manner., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Autophagy dysfunction has been closely related with pathogenesis of many neurodegenerative diseases and therefore represents a potential therapeutic target. Extracellular vesicles (EVs) may act as potent anti-inflammatory agents and also modulators of autophagy in target cells. However, the molecular mechanisms by which EVs modulate autophagy flux in human microglia remain largely unexplored. In the present study, we investigated the effects of EVs derived from human oral mucosa stem cells on the autophagy in human microglia. We demonstrate that EVs promoted autophagy and autophagic flux in human microglia and that this process was dependent on the integrity of lipid rafts. Lipopolysaccharide (LPS) also activated autophagy, but combined treatment with EVs and LPS suppressed autophagy response, indicating interference between these signaling pathways. Blockage of Toll-like receptor 4 (TLR4) with anti-TLR4 antibody suppressed EV-induced autophagy. Furthermore, inhibition of the EV-associated heat shock protein (HSP70) chaperone which is one of the endogenous ligands of the TLR4 also suppressed EV-induced lipid raft formation and autophagy. Pre-treatment of microglia with a selective inhibitor of αvβ3/αvβ5 integrins cilengitide inhibited EV-induced autophagy. Finally, blockage of purinergic P2X4 receptor (P2X4R) with selective inhibitor 5-BDBD also suppressed EV-induced autophagy. In conclusion, we demonstrate that EVs activate autophagy in human microglia through interaction with HSP70/TLR4, αVβ3/αVβ5, and P2X4R signaling pathways and that these effects depend on the integrity of lipid rafts. Our findings could be used to develop new therapeutic strategies targeting disease-associated microglia., (© 2024 Federation of European Biochemical Societies.)

Biological membranes are partitioned into functional zones termed membrane microdomains, which contain specific lipids and proteins 1-3 . 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 behaviour 3,4 . 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 factors 5-9 . 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)P 2 , 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., (© 2024. The Author(s).)

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.