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Cellular & Molecular Biology Letters Oct 2023Membrane rafts play a crucial role in the regulation of many important biological processes. Our previous data suggest that specific interactions of flotillins with MPP1...
BACKGROUND
Membrane rafts play a crucial role in the regulation of many important biological processes. Our previous data suggest that specific interactions of flotillins with MPP1 are responsible for membrane raft domain organization and regulation in erythroid cells. Interaction of the flotillin-based protein network with specific membrane components underlies the mechanism of raft domain formation and regulation, including in cells with low expression of MPP1.
METHODS
We sought to identify other flotillin partners via the immobilized recombinant flotillin-2-based affinity approach and mass spectrometry technique. The results were further confirmed via immunoblotting and via co-immunoprecipitation. In order to study the effect of the candidate protein on the physicochemical properties of the plasma membrane, the gene was knocked down via siRNA, and fluorescence lifetime imaging microscopy and spot-variation fluorescence correlation spectroscopy was employed.
RESULTS
EFR3A was identified as a candidate protein that interacts with flotillin-2. Moreover, this newly discovered interaction was demonstrated via overlay assay using recombinant EFR3A and flotillin-2. EFR3A is a stable component of the detergent-resistant membrane fraction of HeLa cells, and its presence was sensitive to the removal of cholesterol. While silencing the EFR3A gene, we observed decreased order of the plasma membrane of living cells or giant plasma membrane vesicles derived from knocked down cells and altered mobility of the raft probe, as indicated via fluorescence lifetime imaging microscopy and spot-variation fluorescence correlation spectroscopy. Moreover, silencing of EFR3A expression was found to disturb epidermal growth factor receptor and phospholipase C gamma phosphorylation and affect epidermal growth factor-dependent cytosolic Ca concentration.
CONCLUSIONS
Altogether, our results suggest hitherto unreported flotillin-2-EFR3A interaction, which might be responsible for membrane raft organization and regulation. This implies participation of this interaction in the regulation of multiple cellular processes, including those connected with cell signaling which points to the possible role in human health, in particular human cancer biology.
Topics: Humans; Cell Membrane; Epidermal Growth Factor; HeLa Cells; Protein Binding; Membrane Microdomains; Adaptor Proteins, Signal Transducing; Membrane Proteins
PubMed: 37880612
DOI: 10.1186/s11658-023-00497-y -
The Journal of Biological Chemistry Dec 2023Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and glycosphingolipids and serve as major platforms for signal transduction. Cell...
Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and glycosphingolipids and serve as major platforms for signal transduction. Cell detachment from the extracellular matrix (ECM) triggers lipid raft disruption and anoikis, which is a barrier for cancer cells to metastasize. Compared to single circulating tumor cells (CTCs), our recent studies have demonstrated that CD44-mediatd cell aggregation enhances the stemness, survival and metastatic ability of aggregated cells. Here, we investigated whether and how lipid rafts are involved in CD44-mediated cell aggregation. We found that cell detachment, which mimics the condition when tumor cells detach from the ECM to metastasize, induced lipid raft disruption in single cells, but lipid raft integrity was maintained in aggregated cells. We further found that lipid raft integrity in aggregated cells was required for Rac1 activation to prevent anoikis. In addition, CD44 and γ-secretase coexisted at lipid rafts in aggregated cells, which promoted CD44 cleavage and generated CD44 intracellular domain (CD44 ICD) to enhance stemness of aggregated cells. Consequently, lipid raft disruption inhibited Rac1 activation, CD44 ICD generation, and metastasis. Our findings reveal two new pathways regulated by CD44-mediated cell aggregation via maintaining lipid raft integrity. These findings also suggest that targeting cell aggregation-mediated pathways could be a novel therapeutic strategy to prevent CTC cluster-initiated metastasis.
Topics: Cell Aggregation; Extracellular Matrix; Membrane Microdomains; Monomeric GTP-Binding Proteins; Signal Transduction; MDA-MB-231 Cells; Humans; Animals; Mice; Cell Line, Tumor; Mice, Inbred BALB C; Hyaluronan Receptors; rac1 GTP-Binding Protein; Anoikis; Enzyme Activation; Neoplasm Metastasis
PubMed: 37866630
DOI: 10.1016/j.jbc.2023.105377 -
Neurologia 2023Rafts are protein-lipid structural nanodomains involved in efficient signal transduction and the modulation of physiological processes of the cell plasma membrane. Raft... (Review)
Review
INTRODUCTION
Rafts are protein-lipid structural nanodomains involved in efficient signal transduction and the modulation of physiological processes of the cell plasma membrane. Raft disruption in the nervous system has been associated with a wide range of disorders.
DEVELOPMENT
We review the concept of rafts, the nervous system processes in which they are involved, and their role in diseases such as Parkinson's disease, Alzheimer disease, and Huntington disease.
CONCLUSIONS
Based on the available evidence, preservation and/or reconstitution of rafts is a promising treatment strategy for a wide range of neurological disorders.
Topics: Humans; Caveolae; Membrane Microdomains; Cholesterol; Cell Membrane; Alzheimer Disease
PubMed: 37858892
DOI: 10.1016/j.nrleng.2023.10.003 -
Biomacromolecules Nov 2023Herein, we report the formation of drug delivery systems from original thermoresponsive block copolymers containing lipid-based segments. Two acrylate monomers derived...
Membrane-Active Thermoresponsive Block Copolymers Containing a Diacylglycerol-Based Segment: RAFT Synthesis, Doxorubicin Encapsulation, and Evaluation of Cytotoxicity against Breast Cancer Cells.
Herein, we report the formation of drug delivery systems from original thermoresponsive block copolymers containing lipid-based segments. Two acrylate monomers derived from palmitic- or oleic-acid-based diacylglycerols (DAGs) were synthesized and polymerized by the reversible addition-fragmentation chain transfer (RAFT) method. Well-defined DAG-based polymers with targeted molar masses and narrow molar mass distributions were next used as macro-chain transfer agents (macro-CTAs) for the polymerization of -isopropylacrylamide (NIPAAm) or -vinylcaprolactam (NVCL). The obtained amphiphilic block copolymers were formed into polymeric nanoparticles (PNPs) with and without encapsulated doxorubicin and characterized. Their biological assessment indicated appropriate cytocompatibility with the representatives of normal cells. Furthermore, compared to the free drug, increased cytotoxicity and apoptosis or necrosis induction in breast cancer cells was documented, including a highly aggressive and invasive triple-negative MDA-MB-231 cell line.
Topics: Humans; Female; Breast Neoplasms; Diglycerides; Doxorubicin; Polymers; Drug Delivery Systems
PubMed: 37842917
DOI: 10.1021/acs.biomac.3c00580 -
Cell Reports Oct 2023Fatty acids have long been considered essential to brain development; however, the involvement of their synthesis in nervous system formation is unclear. We generate...
Fatty acids have long been considered essential to brain development; however, the involvement of their synthesis in nervous system formation is unclear. We generate mice with knockout of GPSN2, an enzyme for synthesis of very-long-chain fatty acids (VLCFAs) and investigate the effects. Both GPSN2 and GPSN2 mice show abnormal neuronal networks as a result of impaired neuronal polarity determination. Lipidomics of GPSN2 embryos reveal that ceramide synthesis is specifically inhibited depending on FA length; namely, VLCFA-containing ceramide is reduced. We demonstrate that lipid rafts are highly enriched in growth cones and that GPSN2 neurons lose gangliosides in their membranes. Application of C24:0 ceramide, but not C16:0 ceramide or C24:0 phosphatidylcholine, to GPSN2 neurons rescues both neuronal polarity determination and lipid-raft density in the growth cone. Taken together, our results indicate that VLCFA synthesis contributes to physiological neuronal development in brain network formation, in particular neuronal polarity determination through the formation of lipid rafts.
Topics: Animals; Mice; Sphingolipids; Ceramides; Fatty Acids; Membrane Microdomains; Neurons
PubMed: 37816355
DOI: 10.1016/j.celrep.2023.113195 -
Virulence Dec 2023is an emerging spore-forming anaerobe that is often observed in -associated inflammatory bowel disease (IBD) exacerbations. Unlike , neither produces toxins nor...
is an emerging spore-forming anaerobe that is often observed in -associated inflammatory bowel disease (IBD) exacerbations. Unlike , neither produces toxins nor possesses toxin-encoding genetic loci, but is commonly found in both intestinal and extra-intestinal infections. Membrane lipid rafts are composed of dynamic assemblies of cholesterol and sphingolipids, allowing bacteria to gain access to cells. However, the direct interaction between and lipid rafts that confers bacteria the ability to disrupt the intestinal barrier and induce pathogenesis remains unclear. In this study, we investigated the associations among nucleotide-binding oligomerization domain containing 2 (NOD2), lipid rafts, and cytotoxicity in -infected gut epithelial cells. Our results revealed that lipid rafts were involved in -induced NOD2 expression and nuclear factor (NF)-κB activation, triggering an inflammatory response. Reducing cholesterol by simvastatin significantly dampened -induced cell death, indicating that the -induced pathogenicity of cells was lipid raft-dependent. These results demonstrate that NOD2 mobilization into membrane rafts in response to -induced cytotoxicity results in aggravated pathogenicity.
Topics: Clostridioides difficile; Clostridium; NF-kappa B; Membrane Microdomains; Cholesterol
PubMed: 37798913
DOI: 10.1080/21505594.2023.2265048 -
Viruses Aug 2023The molecular mechanisms controlling the adaptation of viruses to host cells are generally poorly documented. An essential issue to resolve is whether host membranes,...
The molecular mechanisms controlling the adaptation of viruses to host cells are generally poorly documented. An essential issue to resolve is whether host membranes, and especially lipid rafts, which are usually considered passive gateways for many enveloped viruses, also encode informational guidelines that could determine virus evolution. Due to their enrichment in gangliosides which confer an electronegative surface potential, lipid rafts impose a first control level favoring the selection of viruses with enhanced cationic areas, as illustrated by SARS-CoV-2 variants. Ganglioside clusters attract viral particles in a dynamic electrostatic funnel, the more cationic viruses of a viral population winning the race. However, electrostatic forces account for only a small part of the energy of raft-virus interaction, which depends mainly on the ability of viruses to form a network of hydrogen bonds with raft gangliosides. This fine tuning of virus-ganglioside interactions, which is essential to stabilize the virus on the host membrane, generates a second level of selection pressure driven by a typical induced-fit mechanism. Gangliosides play an active role in this process, wrapping around the virus spikes through a dynamic quicksand-like mechanism. Viruses are thus in an endless race for access to lipid rafts, and they are bound to evolve perpetually, combining speed (electrostatic potential) and precision (fine tuning of amino acids) under the selective pressure of the immune system. Deciphering the host membrane guidelines controlling virus evolution mechanisms may open new avenues for the design of innovative antivirals.
Topics: Humans; COVID-19; SARS-CoV-2; Antiviral Agents; Gangliosides
PubMed: 37766261
DOI: 10.3390/v15091854 -
Molecules (Basel, Switzerland) Sep 2023The plasma membrane lipid rafts are cholesterol- and sphingolipid-enriched domains that allow regularly distributed, sub-micro-sized structures englobing proteins to... (Review)
Review
The plasma membrane lipid rafts are cholesterol- and sphingolipid-enriched domains that allow regularly distributed, sub-micro-sized structures englobing proteins to compartmentalize cellular processes. These membrane domains can be highly heterogeneous and dynamic, functioning as signal transduction platforms that amplify the local concentrations and signaling of individual components. Moreover, they participate in cell signaling routes that are known to be important targets of environmental toxicants affecting cell redox status and calcium homeostasis, immune regulation, and hormonal functions. In this work, the evidence that plasma membrane raft-like domains operate as hubs for toxicants' cellular actions is discussed, and suggestions for future research are provided. Several studies address the insertion of pesticides and other organic pollutants into membranes, their accumulation in lipid rafts, or lipid rafts' disruption by polychlorinated biphenyls (PCBs), benzo[a]pyrene (B[a]P), and even metals/metalloids. In hepatocytes, macrophages, or neurons, B[a]P, airborne particulate matter, and other toxicants caused rafts' protein and lipid remodeling, oxidative changes, or amyloidogenesis. Different studies investigated the role of the invaginated lipid rafts present in endothelial cells in mediating the vascular inflammatory effects of PCBs. Furthermore, in vitro and in vivo data strongly implicate raft-localized NADPH oxidases, the aryl hydrocarbon receptor, caveolin-1, and protein kinases in the toxic mechanisms of occupational and environmental chemicals.
PubMed: 37764374
DOI: 10.3390/molecules28186598 -
Biomedicines Sep 2023The excess microvascular endothelial permeability is a hallmark of acute inflammatory diseases. Maintenance of microvascular integrity is critical to preventing leakage...
Non-Lethal Doses of RSL3 Impair Microvascular Endothelial Barrier through Degradation of Sphingosie-1-Phosphate Receptor 1 and Cytoskeletal Arrangement in A Ferroptosis-Independent Manner.
The excess microvascular endothelial permeability is a hallmark of acute inflammatory diseases. Maintenance of microvascular integrity is critical to preventing leakage of vascular components into the surrounding tissues. Sphingosine-1-phosphate (S1P) is an active lysophospholipid that enhances the endothelial cell (EC) barrier via activation of its receptor S1PR1. Here, we delineate the effect of non-lethal doses of RSL3, an inhibitor of glutathione peroxidase 4 (GPX4), on EC barrier function. Low doses of RSL3 (50-100 nM) attenuated S1P-induced human lung microvascular barrier enhancement and the phosphorylation of AKT. To investigate the molecular mechanisms by which RSL3 attenuates S1P's effect, we examined the S1PR1 levels. RSL3 treatment reduced S1PR1 levels in 1 h, whereas the effect was attenuated by the proteasome and lysosome inhibitors as well as a lipid raft inhibitor. Immunofluorescence staining showed that RSL3 induced S1PR1 internalization from the plasma membrane into the cytoplasm. Furthermore, we found that RSL3 (100 and 200 nM) increased EC barrier permeability and cytoskeletal rearrangement without altering cell viability. Taken together, our data delineates that non-lethal doses of RSL3 impair EC barrier function via two mechanisms. RSL3 attenuates S1P1-induced EC barrier enhancement and disrupts EC barrier integrity through the generation of 4-hydroxynonena (4HNE). All these effects are independent of ferroptosis.
PubMed: 37760892
DOI: 10.3390/biomedicines11092451 -
BioRxiv : the Preprint Server For... Sep 2023The peroxidation of membrane lipids by free radicals contributes to aging, numerous diseases, and ferroptosis, an iron-dependent form of cell death. Peroxidation changes...
The peroxidation of membrane lipids by free radicals contributes to aging, numerous diseases, and ferroptosis, an iron-dependent form of cell death. Peroxidation changes the structure, conformation and physicochemical properties of lipids, leading to major membrane alterations including bilayer thinning, altered fluidity, and increased permeability. Whether and how lipid peroxidation impacts the lateral organization of proteins and lipids in biological membranes, however, remains poorly understood. Here, we employ cell-derived giant plasma membrane vesicles (GPMVs) as a model to investigate the impact of lipid peroxidation on ordered membrane domains, often termed membrane rafts. We show that lipid peroxidation induced by the Fenton reaction dramatically enhances phase separation propensity of GPMVs into co-existing liquid ordered (raft) and liquid disordered (non-raft) domains and increases the relative abundance of the disordered, non-raft phase. Peroxidation also leads to preferential accumulation of peroxidized lipids and 4-hydroxynonenal (4-HNE) adducts in the disordered phase, decreased lipid packing in both raft and non-raft domains, and translocation of multiple classes of proteins out of rafts. These findings indicate that peroxidation of plasma membrane lipids disturbs many aspects of membrane rafts, including their stability, abundance, packing, and protein and lipid composition. We propose that these disruptions contribute to the pathological consequences of lipid peroxidation during aging and disease, and thus serve as potential targets for therapeutic intervention.
PubMed: 37745342
DOI: 10.1101/2023.09.12.557355