Imaging cholesterol depletion at the plasma membrane by methyl-β-cyclodextrin.
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ABSTRACT:
SUBMITTER: Abe M
PROVIDER: S-EPMC8281586 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Sphingolipid microdomains are thought to result from the organization of plasma membrane sphingolipids and cholesterol into a liquid ordered phase, wherein the glycosylphosphatidylinositol (GPI)-anchored proteins are enriched. These domains, resistant to extraction by cold Triton X-100, can be isolated as buoyant membrane complexes (detergent-resistant membranes) in isopycnic density gradients. Here the effects of methyl-beta-cyclodextrin (MBCD), a specific cholesterol-binding agent that neither binds nor inserts into the plasma membrane, were investigated on the sphingolipid microdomains of lymphocytes. MBCD released substantial quantities of GPI-anchored Thy-1 and glycosphingolipid GM1, and also other surface proteins including CD45, and intracellular Lck and Fyn kinases. From endothelial cells, MBCD released GPI-anchored CD59, and CD44, but only a negligible amount of caveolin. Most MBCD-released Thy-1 and CD59 were not sedimentable and thus differed from Thy-1 released by membrane-active cholesterol-binding agents such as saponin and streptolysin O, or Triton X-100. Unlike that released by Triton X-100, only part of the Thy-1 molecules released by MBCD was buoyant in density gradients and co-isolated with GM1. Finally, treatment of Triton X-100-isolated detergent-resistant membranes with MBCD extracted most of the cholesterol without affecting the buoyant properties of Thy-1 or GM1. We suggest that (1) MBCD preferentially extracts cholesterol from outside, rather than within the sphingolipid microdomains and (2) this partly solubilizes GPI-anchored and transmembrane proteins from the glycerophospholipid-rich membrane and releases sphingolipid microdomains in both vesicular and non-vesicular form.
| S-EPMC1219799 | biostudies-other
Project description:Lipid rafts are cholesterol-rich cell signaling platforms and their physiological role can be explored by cholesterol depletion. To dress a global picture of transcriptional changes ongoing after lipid raft disruption, we performed whole-genome expression profiling in epidermal keratinocytes, a cell type which synthesizes its cholesterol in situ. We used microarrays to identify transcriptional changes in gene expression of cholesterol-depleted keratinocytes. Cholesterol depletion by methyl-beta-cyclodextrin disrupts the organization of lipid rafts, which are cholesterol- and sphingolipid-rich membrane microdomains. Transcript levels were measured in autocrine confluent cultures of normal human epidermal keratinocytes were either left untreated (Ctrl), cholesterol-depleted for 1h with 7.5mM methyl-beta-cyclodextrin (MBCD), or mock cholesterol-depleted for 1h with 7.5mM cholesterol-charged methyl-beta-cyclodextrin complexes (MBCD/chol) (Mock cholesterol depletion is a suppementary negative control as this treatment does not extract cholesterol from cell membranes). Samples are analysed either immediately after the treatment (R0h) or after recovery times of 1h (R1h) respectively 8h (R8h). in total 9 samples are analysed and no replicates are performed.
2010-04-17 | E-GEOD-21364 | biostudies-arrayexpress
Project description:Lipid rafts are cholesterol-rich cell signaling platforms and their physiological role can be explored by cholesterol depletion. To dress a global picture of transcriptional changes ongoing after lipid raft disruption, we performed whole-genome expression profiling in epidermal keratinocytes, a cell type which synthesizes its cholesterol in situ. We used microarrays to identify transcriptional changes in gene expression of cholesterol-depleted keratinocytes. Cholesterol depletion by methyl-beta-cyclodextrin disrupts the organization of lipid rafts, which are cholesterol- and sphingolipid-rich membrane microdomains.
2010-04-17 | GSE21364 | GEO
Project description:Glycosylphosphatidylinositol-linked and transmembrane major histocompatibility complex (MHC) class II I-E(k) proteins, as well as N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Tritc-DHPE), are used as probes to determine the effect of cholesterol concentration on the organization of the plasma membrane at temperatures in the range 22 degrees C-42 degrees C. Cholesterol depletion caused a decrease in the diffusion coefficients for the MHC II proteins and also for a slow fraction of the Tritc-DHPE population. At 37 degrees C, reduction of the total cell cholesterol concentration results in a smaller suppression of the translational diffusion for I-E(k) proteins (twofold) than was observed in earlier work at 22 degrees C (five sevenfold) Vrljic, M., S. Y. Nishimura, W. E. Moerner, and H. M. McConnell. 2005. Biophys. J. 88:334-347. At 37 degrees C, the diffusion of both I-E(k) proteins is Brownian (0.9 < alpha-parameter < 1.1). More than 99% of the protein population diffuses homogeneously when imaged at 65 frames per s. As the temperature is raised from 22 degrees C to 42 degrees C, a change in activation energy is seen at approximately 35 degrees C in the Arrhenius plots. Cytoskeletal effects appear to be minimal. These results are consistent with a previously described model of solid-like domain formation in the plasma membrane.
| S-EPMC1367117 | biostudies-literature
Project description:BackgroundDespite modern advances in treatment, skin cancer is still one of the most common causes of death in the western countries. Chemotherapy plays an important role in melanoma management. Tamoxifen has been used either alone or in- combination with other chemotherapeutic agents to treat melanoma. However, response rate of tamoxifen as a single agent has been comparatively low. In the present study, we investigated whether treatment with methyl-?-cyclodextrin (MCD), a cholesterol depleting agent, increases the efficacy of tamoxifen in melanoma cells.MethodsThis was a two-part study that incorporated in vitro effects of tamoxifen and MCD combination by analyzing cell survival, apoptosis and cell cycle analysis and in vivo antitumor efficacy on tumor isografts in C57BL/6J mice.ResultsMCD potentiated tamoxifen induced anticancer effects by causing cell cycle arrest and induction of apoptosis. Sensitization to tamoxifen was associated with down regulation of antiapoptotic protein Bcl-2, up-regulation of proapoptotic protein Bax, reduced caveolin-1 (Cav-1) and decreased pAkt/pERK levels. Co-administration of tamoxifen and MCD caused significant reduction in tumor volume and tumor weight in mice due to enhancement of drug uptake in the tumor. Supplementation with cholesterol abrogated combined effect of tamoxifen and MCD.ConclusionOur results emphasize a potential synergistic effect of tamoxifen with MCD, and therefore, may provide a unique therapeutic window for improvement in melanoma treatment.
| S-EPMC4175626 | biostudies-literature
Project description:A procedure has been developed that allows for the depletion of rat liver plasma membrane cholesterol by incubation with liposomes at 4 degrees C. Upon cholesterol depletion, adenylate cyclase activity was inhibited and the membranes became more rigid, as determined by the flexibility of an incorporated fatty acid spin probe. Decreasing the cholesterol/phospholipid molar ratio elicited a pronounced drop in the net fold-stimulation of adenylate cyclase activity by glucagon. Two lipid phase separations were detected in cholesterol-depleted membranes at around 25 degrees C and 13 degrees C respectively. Breaks at these temperatures were observed in Arrhenius plots of both the mobility of the spin probe and the glucagon-stimulated adenylate cyclase activity for the range 2-40 degrees C, but only the one at the lower temperature for the fluoride-stimulated activity. It is proposed that the lipid phase separation occurring at 25 degrees C is localized in the external half of the bilayer, whereas that at 13 degrees C is due to lipids in the inner half of the bilayer. Similar structural and functional perturbations were manifest if the cholesterol-complexing polyene antibiotic amphotericin B was added to native membranes. The mechanism of adenylate cyclase inhibition achieved by cholesterol depletion and the domain structure of the plasma membrane in relation to cholesterol distribution are discussed. Native cholesterol/phospholipid ratios appear to optimize the functioning of adenylate cyclase in liver plasma membranes.
| S-EPMC1152051 | biostudies-other
Project description:ObjectiveDysregulation of cholesterol metabolism in the brain is responsible for many lipid storage disorders, including Niemann-Pick disease type C (NPC). Here, we have investigated whether cyclodextrin (CD) and apolipoprotein A-I (apoA-I) induce the same signal to inhibit cell cholesterol accumulation by focusing on the main proteins involved in cholesterol homeostasis in response to CD and apoA-I treatment.Materials and methodsIn this experimental study, astrocytes were treated with apoA-I or CD and then lysed in RIPA buffer. We used Western blot to detect protein levels of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) and ATP-binding cassette transporter A1 (ABCA1). Cell cholesterol content and cholesterol release in the medium were also measured.ResultsApoA-I induced a significant increase in ABCA1 and a mild increase in HMGCR protein level, whereas CD caused a significant increase in HMGCR with a significant decrease in ABCA1. Both apoA-I and CD increased cholesterol release in the medium. A mild, but not significant increase, in cell cholesterol content was seen by apoA-I; however, a significant increase in cell cholesterol was detected when the astrocytes were treated with CD.ConclusionCD, like apoA-I, depletes cellular cholesterol. This depletion occurs in a different way from apoA-I that is through cholesterol efflux. Depletion of cell cholesterol with CDs led to reduced protein levels of ABCA1 along with increased HMGCR and accumulation of cell cholesterol. This suggested that CDs, unlike apoA-I, could impair the balance between cholesterol synthesis and release, and interfere with cellular function that depends on ABCA1.
| S-EPMC7944131 | biostudies-literature
Project description:The cholesterol chelating agent, methyl-?-cyclodextrin (M?CD), alters synaptic function in many systems. At crayfish neuromuscular junctions, M?CD is reported to reduce excitatory junctional potentials (EJPs) by impairing impulse propagation to synaptic terminals, and to have no postsynaptic effects. We examined the degree to which physiological effects of M?CD correlate with its ability to reduce cholesterol, and used thermal acclimatization as an alternative method to modify cholesterol levels. M?CD impaired impulse propagation and decreased EJP amplitude by 40% (P<0.05) in preparations from crayfish acclimatized to 14 °C but not from those acclimatized to 21 °C. The reduction in EJP amplitude in the cold-acclimatized group was associated with a 49% reduction in quantal content (P<0.05). M?CD had no effect on input resistance in muscle fibers but decreased sensitivity to the neurotransmitter L-glutamate in both warm- and cold-acclimatized groups. This effect was less pronounced and reversible in the warm-acclimatized group (90% reduction in cold, P<0.05; 50% reduction in warm, P<0.05). M?CD reduced cholesterol in isolated nerve and muscle from cold- and warm-acclimatized groups by comparable amounts (nerve: 29% cold, 25% warm; muscle: 20% cold, 18% warm; P<0.05). This effect was reversed by cholesterol loading, but only in the warm-acclimatized group. Thus, effects of M?CD on glutamate-sensitivity correlated with its ability to reduce cholesterol, but effects on impulse propagation and resulting EJP amplitude did not. Our results indicate that M?CD can affect both presynaptic and postsynaptic properties, and that some effects of M?CD are unrelated to cholesterol chelation.
| S-EPMC3348160 | biostudies-literature
Project description:Adipocytes store a significant amount of cholesterol and triglycerides. However, whether cholesterol modulates adipocyte function remains largely unknown. We modulated the cholesterol level in adipocytes to examine its effect on the secretion of adiponectin, an important hormone specifically secreted by adipocytes. Treating differentiated 3T3-L1 adipocytes with 4 mM methyl-β-cyclodextrin (MβCD), a molecule with a high affinity for cholesterol, rapidly depleted cholesterol in adipocytes. Interestingly, MβCD treatment increased adiponectin in the medium without affecting its intracellular level, suggesting a modulation of secretion. By contrast, cholesterol addition did not affect adiponectin secretion, suggesting that cholesterol-depletion-induced intracellular cholesterol trafficking, but not reduced cholesterol level, accounted for MβCD-induced adiponectin secretion. MβCD-induced adiponectin secretion was reduced after 10 μg/mL U18666A treatment that suppressed cholesterol transport out of late endosomes/lysosomes. Depleting Niemann-Pick type C1 (NPC1) or NPC2 proteins, which mediate endosomal/lysosomal cholesterol export, consistently reduced MβCD-induced adiponectin secretion. Furthermore, treatment with 1 μM bafilomycin A1, which neutralized acidic endosomes/lysosomes, also attenuated MβCD-induced adiponectin secretion. Finally, MβCD treatment redistributed cellular adiponectin to lower-density fractions in sucrose gradient fractionation. Our results show that MβCD-mediated cholesterol depletion elevates the secretion of adiponectin, highlighting the involvement of endosomes and lysosomes in adiponectin secretion in adipocytes.
| S-EPMC10572842 | biostudies-literature
Project description:Recent results provide evidence that cholesterol is highly accessible for removal from both cell and model membranes above a threshold concentration that varies with membrane composition. Here we measured the rate at which methyl-?-cyclodextrin depletes cholesterol from a supported lipid bilayer as a function of cholesterol mole fraction. We formed supported bilayers from two-component mixtures of cholesterol and a PC (phosphatidylcholine) lipid, and we directly visualized the rate of decrease in area of the bilayers with fluorescence microscopy. Our technique yields the accessibility of cholesterol over a wide range of concentrations (30-66 mol %) for many individual bilayers, enabling fast acquisition of replicate data. We found that the bilayers contain two populations of cholesterol, one with low surface accessibility and the other with high accessibility. A larger fraction of the total membrane cholesterol appears in the more accessible population when the acyl chains of the PC-lipid tails are more unsaturated. Our findings are most consistent with the predictions of the condensed-complex and cholesterol bilayer domain models of cholesterol-phospholipid interactions in lipid membranes.
| S-EPMC4744159 | biostudies-literature