Cholesterol-enriched Microdomains Research Articles

Low Density Lipoprotein Cholesterol Decreases the Expression of Adenosine A2A Receptor and Lipid Rafts-Protein Flotillin-1: Insights on Cardiovascular Risk of Hypercholesterolemia.

High blood levels of low-density lipoprotein (LDL)-cholesterol (LDL-C) are associated with atherosclerosis, mainly by promoting foam cell accumulation in vessels. As cholesterol is an essential component of cell plasma membranes and a regulator of several signaling pathways, LDL-C excess may have wider cardiovascular toxicity. We examined, in untreated hypercholesterolemia (HC) patients, selected regardless of the cause of LDL-C accumulation, and in healthy participants (HP), the expression of the adenosine A2A receptor (A2AR), an anti-inflammatory and vasodilatory protein with cholesterol-dependent modulation, and Flotillin-1, protein marker of cholesterol-enriched plasma membrane domains. Blood cardiovascular risk and inflammatory biomarkers were measured. A2AR and Flotillin-1 expression in peripheral blood mononuclear cells (PBMC) was lower in patients compared to HP and negatively correlated to LDL-C blood levels. No other differences were observed between the two groups apart from transferrin and ferritin concentrations. A2AR and Flotillin-1 proteins levels were positively correlated in the whole study population. Incubation of HP PBMCs with LDL-C caused a similar reduction in A2AR and Flotillin-1 expression. We suggest that LDL-C affects A2AR expression by impacting cholesterol-enriched membrane microdomains. Our results provide new insights into the molecular mechanisms underlying cholesterol toxicity, and may have important clinical implication for assessment and treatment of cardiovascular risk in HC.

Shear Stress and Sub-Femtomolar Levels of Ligand Synergize to Activate ALK1 Signaling in Endothelial Cells.

Endothelial cells (ECs) respond to concurrent stimulation by biochemical factors and wall shear stress (SS) exerted by blood flow. Disruptions in flow-induced responses can result in remodeling issues and cardiovascular diseases, but the detailed mechanisms linking flow-mechanical cues and biochemical signaling remain unclear. Activin receptor-like kinase 1 (ALK1) integrates SS and ALK1-ligand cues in ECs; ALK1 mutations cause hereditary hemorrhagic telangiectasia (HHT), marked by arteriovenous malformation (AVM) development. However, the mechanistic underpinnings of ALK1 signaling modulation by fluid flow and the link to AVMs remain uncertain. We recorded EC responses under varying SS magnitudes and ALK1 ligand concentrations by assaying pSMAD1/5/9 nuclear localization using a custom multi-SS microfluidic device and a custom image analysis pipeline. We extended the previously reported synergy between SS and BMP9 to include BMP10 and BMP9/10. Moreover, we demonstrated that this synergy is effective even at extremely low SS magnitudes (0.4 dyn/cm2) and ALK1 ligand range (femtogram/mL). The synergistic response to ALK1 ligands and SS requires the kinase activity of ALK1. Moreover, ALK1's basal activity and response to minimal ligand levels depend on endocytosis, distinct from cell-cell junctions, cytoskeleton-mediated mechanosensing, or cholesterol-enriched microdomains. However, an in-depth analysis of ALK1 receptor trafficking's molecular mechanisms requires further investigation.

Lipidation states orchestrate CLICK-III/CaMKIγ's stepwise association with Golgi and rafts-enriched membranes and specify its functional coupling to STEF-Rac1-dependent neurite extension.

CLICK-III/CaMKIγ is a lipid-anchored neuronal isoform of multifunctional Ca2+/calmodulin-dependent protein kinases, which mediates BDNF-dependent dendritogenesis in cultured cortical neurons. We found that two distinct lipidation states of CaMKIγ, namely, prenylation and palmitoylation, controlled its association with detergent-resistant microdomains in the dendrites and were essential for its dendritogenic activity. However, the impact of each lipid modification on membrane targeting/trafficking and how it specifies functional coupling leading to polarized changes in neuronal morphology are not clear. Here, we show that prenylation induces membrane anchoring of CaMKIγ, permitting access to the Golgi apparatus, and a subsequent palmitoylation facilitates association with cholesterol-enriched lipid microdomains or lipid rafts, in particular at the Golgi. To specifically test the role of palmitoylated CaMKγ in neurite extension, we identified and took advantage of a cell system, PC12, which, unlike neurons, conveniently lacked CaMKIγ and was deficient in the activity-dependent release of a neuritogenic growth factor while possessing the ability to activate polarized rafts signaling for morphogenesis. This system allowed us to rigorously demonstrate that an activity-dependent, lipid rafts-restricted Rac activation leading to neuritogenesis could be functionally rescued by dually lipidated CaMKIγ expression, revealing that not only prenylation but also palmitoylation is essential for CaMKIγ to activate a compartmentalized STEF-Rac1 pathway. These results shed light on the significance of recruiting prenylated and palmitoylated CaMKIγ into the coalescing signalosomes at lipid rafts together with Rac1 and its specific GEF and STEF and forming a compartmentalized Ca2+ signaling pathway that underlies activity-dependent neuritogenesis and morphogenesis during axodendritic polarization critical for brain development and circuitogenesis.

Viral modulation of lipid rafts and their potential as putative antiviral targets.

Lipid rafts are ubiquitous in cells. They are identified as cholesterol and glycosphingolipid enriched microdomains on cellular membranes. They serve as platforms for cellular communications by functioning in signal transduction and membrane trafficking. Such structural organisation fulfils cellular needs for normal function, but at the same time increases vulnerability of cells to pathogen invasion. Viruses rely heavily on lipid rafts in basically every stage of the viral life cycle for successful infection. Various mechanisms of lipid rafts modification exploited by diverse viruses for attachment, internalisation, membrane fusion, genome replication, assembly and release have been brought to light. This review focuses on virus-raft interactions and how a wide range of viruses manipulate lipid rafts at distinct stages of infection. The importance of virus-raft interactions in viral infections has inspired researchers to discover and develop antivirals that target this interaction, such as statins, methyl-β-cyclodextrin, viperin, 25-hydroxycholesterol and even anti-malarial drugs. The therapeutic modulations of lipid rafts as potential antiviral intervention from in vitro and in vivo evidence are discussed herein.

Membrane lipid rafts are required for AMPA receptor tyrosine phosphorylation.

Membrane lipid rafts are sphingolipids and cholesterol-enriched membrane microdomains, which form a center for the interaction or assembly of palmitoylated signaling molecules, including Src family non-receptor type protein tyrosine kinases. Lipid rafts abundantly exist in neurons and function in the maintenance of synapses. Excitatory synaptic strength is largely controlled by the surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors in the mammalian brain. AMPA receptor endocytosis from the synaptic surface is regulated by phosphorylation of the GluA2 subunit at tyrosine 876 by Src family kinases. Here, I revealed that tyrosine phosphorylated GluA2 is concentrated in the lipid rafts fraction. Furthermore, stimulation-induced upregulation of GluA2 tyrosine phosphorylation is disrupted by the treatment of neurons with a cholesterol-depleting compound, filipin III. These results indicate the importance of lipid rafts as enzymatic reactive sites for AMPA receptor tyrosine phosphorylation and subsequent AMPA receptor internalization from the synaptic surface.

SNAP23-Mediated Perturbation of Cholesterol-Enriched Membrane Microdomain Promotes Extracellular Vesicle Production in Src-Activated Cancer Cells.

Extracellular vesicles (EVs) originating from intraluminal vesicles (ILVs) formed within multivesicular bodies (MVBs), often referred to as small EV (sEV) or exosomes, are aberrantly produced by cancer cells and regulate the tumor microenvironment. The tyrosine kinase c-Src is upregulated in a wide variety of human cancers and is involved in promoting sEV secretion, suggesting its role in malignant progression. In this study, we found that activated Src liberated synaptosomal-associated protein 23 (SNAP23), a SNARE molecule, from lipid rafts to non-rafts on cellular membrane. We also demonstrated that SNAP23 localized in non-rafts induced cholesterol downregulation and ILV formation, resulting in the upregulation of sEV production in c-Src-transformed cells. Furthermore, the contribution of the SNAP23-cholesterol axis on sEV upregulation was confirmed in pancreatic cancer cells. High SNAP23 expression is associated with poor prognosis in patients with pancreatic cancer. These findings suggest a unique mechanism for the upregulation of sEV production via SNAP23-mediated cholesterol downregulation in Src-activated cancer cells.

Different Membrane Environments Generate Multiple Functions of P-type Ion Pumps

P-type ion pumps (P-type ATPases) are involved in various fundamental biological processes. For example, the gastric proton pump (H+,K+-ATPase) and sodium pump (Na+,K+-ATPase) are responsible for secretion of gastric acid and maintenance of cell membrane potential, respectively. In this review, we summarize three topics of our studies. The first topic is gastric H+,K+-ATPase associated with Cl--transporting proteins (Cl-/H+ exchanger ClC-5 and K+-Cl- cotransporter KCC4). In gastric parietal cells, we found that ClC-5 is predominantly expressed in intracellular tubulovesicles and that KCC4 is predominantly expressed in the apical membrane. Gastric acid (HCl) secretion may be accomplished by the two different complexes of H+,K+-ATPase and Cl--transporting protein. The second topic focuses on the Na+,K+-ATPase α1-isoform (α1NaK) associated with the volume-regulated anion channel (VRAC). In the cholesterol-enriched membrane microdomains of human cancer cells, we found that α1NaK has a receptor-like (non-pumping) function and that binding of low concentrations (nM level) of cardiac glycosides to α1NaK activates VRAC and exerts anti-cancer effects without affecting the pumping function of α1NaK. The third topic is the Na+,K+-ATPase α3-isoform (α3NaK) in human cancer cells. We found that α3NaK is abnormally expressed in the intracellular vesicles of attached cancer cells and that the plasma membrane translocation of α3NaK upon cell detachment contributes to the survival of metastatic cancer cells. Our results indicate that multiple functions of P-type ion pumps are generated by different membrane environments and their associated proteins.

Autophagic Activation and Decrease of Plasma Membrane Cholesterol Contribute to Anticancer Activities in Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC), an aggressive subtype of pulmonary carcinomas with high mortality, accounts for 85% of all lung cancers. Drug resistance and high recurrence rates impede the chemotherapeutic effect, making it urgent to develop new anti-NSCLC agents. Recently, we have demonstrated that para-toluenesulfonamide is a potential anti-tumor agent in human castration-resistant prostate cancer (CRPC) through inhibition of Akt/mTOR/p70S6 kinase pathway and lipid raft disruption. In the current study, we further addressed the critical role of cholesterol-enriched membrane microdomain and autophagic activation to para-toluenesulfonamide action in killing NSCLC. Similar in CRPC, para-toluenesulfonamide inhibited the Akt/mTOR/p70S6K pathway in NSCLC cell lines NCI-H460 and A549, leading to G1 arrest of the cell cycle and apoptosis. Para-toluenesulfonamide significantly decreased the cholesterol levels of plasma membrane. External cholesterol supplement rescued para-toluenesulfonamide-mediated effects. Para-toluenesulfonamide induced a profound increase of LC3-II protein expression and a significant decrease of p62 expression. Double staining of lysosomes and cellular cholesterol showed para-toluenesulfonamide-induced lysosomal transportation of cholesterol, which was validated using flow cytometric analysis of lysosome staining. Moreover, autophagy inhibitors could blunt para-toluenesulfonamide-induced effect, indicating autophagy induction. In conclusion, the data suggest that para-toluenesulfonamide is an effective anticancer agent against NSCLC through G1 checkpoint arrest and apoptotic cell death. The disturbance of membrane cholesterol levels and autophagic activation may play a crucial role to para-toluenesulfonamide action.

The role of sigma 1 receptor in organization of endoplasmic reticulum signaling microdomains.

Sigma 1 receptor (S1R) is a 223-amino-acid-long transmembrane endoplasmic reticulum (ER) protein. S1R modulates activity of multiple effector proteins and is a well-established drug target. However, signaling functions of S1R in cells are poorly understood. Here, we test the hypothesis that biological activity of S1R in cells can be explained by its ability to interact with cholesterol and to form cholesterol-enriched microdomains in the ER membrane. By performing experiments in reduced reconstitution systems, we demonstrate direct effects of cholesterol on S1R clustering. We identify a novel cholesterol-binding motif in the transmembrane region of human S1R. Mutations of this motif impair association of recombinant S1R with cholesterol beads, affect S1R clustering in vitro and disrupt S1R subcellular localization. We demonstrate that S1R-induced membrane microdomains have increased local membrane thickness and that increased local cholesterol concentration and/or membrane thickness in these microdomains can modulate signaling of inositol-requiring enzyme 1α in the ER. Further, S1R agonists cause disruption of S1R clusters, suggesting that biological activity of S1R agonists is linked to remodeling of ER membrane microdomains. Our results provide novel insights into S1R-mediated signaling mechanisms in cells.

Sigma-1 Receptor (S1R) Interaction with Cholesterol: Mechanisms of S1R Activation and Its Role in Neurodegenerative Diseases.

The sigma-1 receptor (S1R) is a 223 amino acid-long transmembrane endoplasmic reticulum (ER) protein. The S1R modulates the activity of multiple effector proteins, but its signaling functions are poorly understood. S1R is associated with cholesterol, and in our recent studies we demonstrated that S1R association with cholesterol induces the formation of S1R clusters. We propose that these S1R-cholesterol interactions enable the formation of cholesterol-enriched microdomains in the ER membrane. We hypothesize that a number of secreted and signaling proteins are recruited and retained in these microdomains. This hypothesis is consistent with the results of an unbiased screen for S1R-interacting partners, which we performed using the engineered ascorbate peroxidase 2 (APEX2) technology. We further propose that S1R agonists enable the disassembly of these cholesterol-enriched microdomains and the release of accumulated proteins such as ion channels, signaling receptors, and trophic factors from the ER. This hypothesis may explain the pleotropic signaling functions of the S1R, consistent with previously observed effects of S1R agonists in various experimental systems.

Novel and Converging Ways of NOX2 and SOD3 in Trafficking and Redox Signaling in Macrophages.

Macrophages and related tissue macrophage populations use the classical NADPH oxidase (NOX2) for the regulated production of superoxide and derived oxidants for pathogen combat and redox signaling. With an emphasis on macrophages, we discuss how sorting into secretory storage vesicles, agonist-responsive membrane trafficking, and segregation into sphingolipid and cholesterol-enriched microdomains (lipid rafts) determine the subcellular distribution and spatial organization of NOX2 and superoxide dismutase-3 (SOD3). We discuss how inflammatory activation of macrophages, in part through small GTPase Rab27A/B regulation of the secretory compartments, mediates the coalescence of these two proteins on the cell surface to deliver a focalized hydrogen peroxide output. In interplay with membrane-embedded oxidant transporters and redox sensitive target proteins, this arrangement allows for the autocrine and paracrine signaling, which govern macrophage activation states and transcriptional programs. By discussing examples of autocrine and paracrine redox signaling, we highlight why formation of spatiotemporal microenvironments where produced superoxide is rapidly converted to hydrogen peroxide and conveyed immediately to reach redox targets in proximal vicinity is required for efficient redox signaling. Finally, we discuss the recent discovery of macrophage-derived exosomes as vehicles of NOX2 holoenzyme export to other cells.

Sigma 1 Receptor, Cholesterol and Endoplasmic Reticulum Contact Sites.

Recent studies indicated potential importance of membrane contact sites (MCS) between the endoplasmic reticulum (ER) and other cellular organelles. These MCS have unique protein and lipid composition and serve as hubs for inter-organelle communication and signaling. Despite extensive investigation of MCS protein composition and functional roles, little is known about the process of MCS formation. In this perspective, we propose a hypothesis that MCS are formed not as a result of random interactions between membranes of ER and other organelles but on the basis of pre-existing cholesterol-enriched ER microdomains.

Preferred Endocytosis of Amyloid Precursor Protein from Cholesterol-Enriched Lipid Raft Microdomains.

Amyloid precursor protein (APP) at the plasma membrane is internalized via endocytosis and delivered to endo/lysosomes, where neurotoxic amyloid-β (Aβ) is produced via β-, γ-secretases. Hence, endocytosis plays a key role in the processing of APP and subsequent Aβ generation. β-, γ-secretases as well as APP are localized in cholesterol-enriched lipid raft microdomains. However, it is still unclear whether lipid rafts are the site where APP undergoes endocytosis and whether cholesterol levels affect this process. In this study, we found that localization of APP in lipid rafts was increased by elevated cholesterol level. We also showed that increasing or decreasing cholesterol levels increased or decreased APP endocytosis, respectively. When we labeled cell surface APP, APP localized in lipid rafts preferentially underwent endocytosis compared to nonraft-localized APP. In addition, APP endocytosis from lipid rafts was regulated by cholesterol levels. Our results demonstrate for the first time that cholesterol levels regulate the localization of APP in lipid rafts affecting raft-dependent APP endocytosis. Thus, regulating the microdomain localization of APP could offer a new therapeutic strategy for Alzheimer’s disease.

Growth cone repulsion to Netrin-1 depends on lipid raft microdomains enriched in UNC5 receptors

During brain development, Uncoordinated locomotion 5 (UNC5) receptors control axonal extension through their sensing of the guidance molecule Netrin-1. The correct positioning of receptors into cholesterol-enriched membrane raft microdomains is crucial for the efficient transduction of the recognized signals. However, whether such microdomains are required for the appropriate axonal guidance mediated by UNC5 receptors remains unknown. Here, we combine the use of confocal microscopy, live-cell FRAP analysis and single-particle tracking PALM to characterize the distribution of UNC5 receptors into raft microdomains, revealing differences in their membrane mobility properties. Using pharmacological and genetic approaches in primary neuronal cultures and brain cerebellar explants we further demonstrate that disrupting raft microdomains inhibits the chemorepulsive response of growth cones and axons against Netrin-1. Together, our findings indicate that the distribution of all UNC5 receptors into cholesterol-enriched raft microdomains is heterogeneous and that the specific localization has functional consequences for the axonal chemorepulsion against Netrin-1.

The Impact of Plasma Membrane Lipid Composition on Flagellum-Mediated Adhesion of Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of foodborne gastrointestinal illness. The adhesion of EHEC to host tissues is the first step enabling bacterial colonization. Adhesins such as fimbriae and flagella mediate this process. Here, we studied the interaction of the bacterial flagellum with the host cell's plasma membrane using giant unilamellar vesicles (GUVs) as a biologically relevant model. Cultured cell lines contain many different molecular components, including proteins and glycoproteins. In contrast, with GUVs, we can characterize the bacterial mode of interaction solely with a defined lipid part of the cell membrane. Bacterial adhesion on GUVs was dependent on the presence of the flagellar filament and its motility. By testing different phospholipid head groups, the nature of the fatty acid chains, or the liposome curvature, we found that lipid packing is a key parameter to enable bacterial adhesion. Using HT-29 cells grown in the presence of polyunsaturated fatty acid (α-linolenic acid) or saturated fatty acid (palmitic acid), we found that α-linolenic acid reduced adhesion of wild-type EHEC but not of a nonflagellated mutant. Finally, our results reveal that the presence of flagella is advantageous for the bacteria to bind to lipid rafts. We speculate that polyunsaturated fatty acids prevent flagellar adhesion on membrane bilayers and play a clear role for optimal host colonization. Flagellum-mediated adhesion to plasma membranes has broad implications for host-pathogen interactions.IMPORTANCE Bacterial adhesion is a crucial step to allow bacteria to colonize their hosts, invade tissues, and form biofilm. Enterohemorrhagic Escherichia coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis. Here, we use biomimetic membrane models and cell lines to decipher the impact of lipid content of the plasma membrane on enterohemorrhagic E. coli flagellum-mediated adhesion. Our findings provide evidence that polyunsaturated fatty acid (α-linolenic acid) inhibits E. coli flagellar adhesion to the plasma membrane in a mechanism separate from its antimicrobial and anti-inflammatory functions. In addition, we confirm that cholesterol-enriched lipid microdomains, often called lipid rafts, are important in bacterial adhesion. These findings demonstrate that plasma membrane adhesion via bacterial flagella play a significant role for an important human pathogen. This mechanism represents a promising target for the development of novel antiadhesion therapies.

Migrasome formation is mediated by assembly of micron-scale tetraspanin macrodomains.

Migrasomes are recently discovered cellular organelles that form as large vesicle-like structures on retraction fibres of migrating cells. While the process of migrasome formation has been described before, the molecular mechanism underlying migrasome biogenesis remains unclear. Here, we propose that the mechanism of migrasome formation consists of the assembly of tetraspanin- and cholesterol-enriched membrane microdomains into micron-scale macrodomains, which swell into migrasomes. The major finding underlying the mechanism is that tetraspanins and cholesterol are necessary and sufficient for migrasome formation. We demonstrate the necessity of tetraspanins and cholesterol via live-cell experiments, and their sufficiency by generating migrasome-like structures in reconstituted membrane systems. We substantiate the mechanism by a theoretical model proposing that the key factor driving migrasome formation is the elevated membrane stiffness of the tetraspanin- and cholesterol-enriched macrodomains. Finally, the theoretical model was quantitatively validated by experimental demonstration of the membrane-stiffening effect of tetraspanin 4 and cholesterol.

Follicle Stimulating Hormone Receptor Signaling is Regulated by Lipid Raft Residency

Infertility affects 7.4 million women and their partners in the United States. One cause for infertility in women is human follicle stimulating hormone (hFSH) dysfunction. hFSH is involved in follicle maturation and estrogen production in females and spermatogenesis in males. Lack of normal hFSH function in either the ligand or receptor leads to infertility in women, however does not fully inhibit fertility in males. hFSH is secreted from the pituitary gland and binds to its g protein‐coupled receptor (hFSHR) located in the ovaries or the testis. Previous research in our lab has shown that hFSHR resides in cholesterol, sphingolipid, and caveolin enriched microdomains called lipid rafts. It has been previously shown that hFSHR interacts with caveolin although the specific mechanism of this interaction is unknown. One possibility is that the interaction is through a specific amino acid sequence in hFSHR consistent with caveolin interaction motifs identified in other proteins. The current study investigated how lipid rafts regulate hFSHR signaling. It was hypothesized that disruption of lipid rafts or interference with the caveolin interaction motif would alter hFSHR signaling. Lipid rafts in human granulosa cells (the hGrC1 cell line) were disrupted through cholesterol depletion with methyl‐β‐cyclodextrin (MβCD) and FSH signaling differences were observed by western blot. It was observed that MβCD treatment resulted in an increase of cAMP signaling compared to control cells. In parallel, mutations in the hFSHR caveolin interaction motif were made and expression plasmids were transiently transfected into HEK 293 cells. Western blot analysis revealed that some of the mutants showed increased basal signaling relative to the wild type receptor. Taken together, these results suggest that lipid rafts may be acting act as an inhibitory mechanism for hFSHR and that caveolin may play a role in receptor recruitment to the lipid raft. Researching hFSH signaling after lipid raft manipulation can provide a better understanding of the cellular mechanisms of hFSHR, which could lead to innovative ideas in infertility treatment for women.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cholesterol-enriched membrane microdomains are needed for insulin signaling and proliferation in hepatic cells.

Hepatocyte proliferation during liver regeneration is a well-coordinated process regulated by the activation of several growth factor receptors, including the insulin receptor (IR). The IR can be localized in part to cholesterol-enriched membrane microdomains, but the role of such domains in insulin-mediated events in hepatocytes is not known. We investigated whether partitioning of IRs into cholesterol-enriched membrane rafts is important for the mitogenic effects of insulin in the hepatic cells. IR and lipid rafts were labeled in HepG2 cells and primary rat hepatocytes. Membrane cholesterol was depleted in vitro with metyl-β-cyclodextrin (MβCD) and in vivo with lovastatin. Insulin-induced calcium (Ca2+) signals studies were examined in HepG2 cells and in freshly isolated rat hepatocytes as well as in whole liver in vivo by intravital confocal imaging. Liver regeneration was studied by 70% partial hepatectomy (PH), and hepatocyte proliferation was assessed by PCNA staining. A subpopulation of IR was found in membrane microdomains enriched in cholesterol. Depletion of cholesterol from plasma membrane resulted in redistribution of the IR along the cells, which was associated with impaired insulin-induced nuclear Ca2+ signals, a signaling event that regulates hepatocyte proliferation. Cholesterol depletion also led to ERK1/2 hyper-phosphorylation. Lovastatin administration to rats decreased hepatic cholesterol content, disrupted lipid rafts and decreased insulin-induced Ca2+ signaling in hepatocytes, and delayed liver regeneration after PH. Therefore, membrane cholesterol content and lipid rafts integrity showed to be important for the proliferative effects of insulin in hepatic cells. NEW & NOTEWORTHY One of insulin's actions is to stimulate liver regeneration. Here we show that a subpopulation of insulin receptors is in a specialized cholesterol-enriched region of the cell membrane and this subfraction is important for insulin's proliferative effects.

Tumor cell cholesterol depletion and V-ATPase inhibition as an inhibitory mechanism to prevent cell migration and invasiveness in melanoma

BackgroundV-ATPase interactions with cholesterol enriched membrane microdomains have been related to metastasis in a variety of cancers, but the underlying mechanism remains at its beginnings. It has recently been reported that the inhibition of this H+ pump affects cholesterol mobilization to the plasma membrane. MethodsInhibition of melanoma cell migration and invasiveness was assessed by wound healing and Transwell assays in murine cell lines (B16F10 and Melan-A). V-ATPase activity was measured in vitro by ATP hydrolysis and H+ transport in membrane vesicles, and intact cell H+ fluxes were measured by using a non-invasive Scanning Ion-selective Electrode Technique (SIET). ResultsCholesterol depletion by 5mM MβCD was found to be inhibitory to the hydrolytic and H+ pumping activities of the V-ATPase of melanoma cell lines, as well as to the migration and invasiveness capacities of these cells. Nearly the same effects were obtained using concanamycin A, a specific inhibitor of V-ATPase, which also promoted a decrease of the H+ efflux in live cells at the same extent of MβCD. ConclusionsWe found that cholesterol depletion significantly affects the V-ATPase activity and the initial metastatic processes following a profile similar to those observed in the presence of the V-ATPase specific inhibitor, concanamycin. General significanceThe results shed new light on the functional role of the interactions between V-ATPases and cholesterol-enriched microdomains of cell membranes that contribute with malignant phenotypes in melanoma.

Membrane cholesterol depletion in cortical neurons highlights altered NMDA receptor functionality in a mouse model of amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a chronic neurodegenerative disease affecting upper and lower motor neurons, with unknown aetiology. Lipid rafts, cholesterol enriched microdomains of the plasma membrane, have been linked to neurodegenerative disorders like ALS. The NMDA-receptor subcellular localization in lipid rafts is known to play many roles, from modulating memory strength to neurotoxicity. In this study, performed on the widely used G93A mouse model of ALS, we have shown an equal content of total membrane cholesterol in Control and G93A cortical cultures. Moreover, by electrophysiological studies, we have recorded NMDA- and AMPA-evoked currents which were not significantly different between the two neuronal populations. To study the role of membrane cholesterol on glutamate receptor functionality, we have analysed NMDA and AMPA receptors following cholesterol membrane depletion by methyl-β-cyclodextrin (MβCD). Interestingly, MβCD chronic treatment has provoked a significant reduction of NMDA-evoked currents in both cellular populations which was dose- and time-dependent but significantly higher in ALS neurons compared to Control. The different MβCD effect on NMDA-evoked currents was not due to a different membrane receptor subunit composition but seemed to cause in both neuronal populations a NMDA receptor membrane redistribution. MβCD treatment effect was receptor-specific since no alterations in the two neuronal populations were detected on AMPA receptors.These results lead us to speculate for an altered proteomic composition of lipid rafts in cortical mutated neurons and suggest the need for further studies on the lipid rafts composition and on their interaction with membrane receptors in ALS cortices.