Depletion Of Membrane Cholesterol Research Articles

Inhibition of CaV2.3 channels by NK1 receptors is sensitive to membrane cholesterol but insensitive to caveolin-1.

Voltage-gated, CaV2.3 calcium channels and neurokinin-1 (NK1) receptors are both present in nuclei of the central nervous system. When transiently coexpressed in human embryonic kidney (HEK) 293 cells, CaV2.3 is primarily inhibited during strong, agonist-dependent activation of NK1 receptors. NK1 receptors localize to plasma membrane rafts, and their modulation by Gq/11 protein-coupled signaling is sensitive to plasma membrane cholesterol. Here, we show that inhibition of CaV2.3 by NK1 receptors is attenuated following methyl-β-cyclodextrin (MBCD)-mediated depletion of membrane cholesterol. By contrast, inhibition of CaV2.3 was unaffected by intracellular diffusion of caveolin-1 scaffolding peptide or by overexpression of caveolin-1. Interestingly, MΒCD treatment had no effect on the macroscopic biophysical properties of CaV2.3, though it significantly decreased whole-cell membrane capacitance. Our data indicate that (1) cholesterol supports at least one component of the NK1 receptor-linked signaling pathway that inhibits CaV2.3 and (2) caveolin-1 is dispensable within this pathway. Our findings suggest that NK1 receptors reside within non-caveolar membrane rafts and that CaV2.3 resides nearby but outside the rafts. Raft-dependent modulation of CaV2.3 could be important in the physiological and pathophysiological processes in which these channels participate, including neuronal excitability, synaptic plasticity, epilepsy, and chronic pain.

Sarcolemmal cholesterol and caveolin-3 dependence of cardiac function, ischemic tolerance, and opioidergic cardioprotection.

Cholesterol-rich caveolar microdomains and associated caveolins influence sarcolemmal ion channel and receptor function and protective stress signaling. However, the importance of membrane cholesterol content to cardiovascular function and myocardial responses to ischemia-reperfusion (I/R) and cardioprotective stimuli are unclear. We assessed the effects of graded cholesterol depletion with methyl-β-cyclodextrin (MβCD) and lifelong knockout (KO) or overexpression (OE) of caveolin-3 (Cav-3) on cardiac function, I/R tolerance, and opioid receptor (OR)-mediated protection. Langendorff-perfused hearts from young male C57Bl/6 mice were untreated or treated with 0.02-1.0 mM MβCD for 25 min to deplete membrane cholesterol and disrupt caveolae. Hearts were subjected to 25-min ischemia/45-min reperfusion, and the cardioprotective effects of morphine applied either acutely or chronically [sustained ligand-activated preconditioning (SLP)] were assessed. MβCD concentration dependently reduced normoxic contractile function and postischemic outcomes in association with graded (10-30%) reductions in sarcolemmal cholesterol. Cardioprotection with acute morphine was abolished with ≥20 μM MβCD, whereas SLP was more robust and only inhibited with ≥200 μM MβCD. Deletion of Cav-3 also reduced, whereas Cav-3 OE improved, myocardial I/R tolerance. Protection via SLP remained equally effective in Cav-3 KO mice and was additive with innate protection arising with Cav-3 OE. These data reveal the membrane cholesterol dependence of normoxic myocardial and coronary function, I/R tolerance, and OR-mediated cardioprotection in murine hearts (all declining with cholesterol depletion). In contrast, baseline function appears insensitive to Cav-3, whereas cardiac I/R tolerance parallels Cav-3 expression. Novel SLP appears unique, being less sensitive to cholesterol depletion than acute OR protection and arising independently of Cav-3 expression.

Cholesterol depletion induces transcriptional changes during skeletal muscle differentiation.

BackgroundMyoblasts undergo major changes in their plasma membrane during the initial steps of skeletal muscle differentiation, including major alterations in the distribution of cholesterol. Cholesterol is involved in crucial membrane functions, such as fluidity, and permeability, and in the organization of specialized membrane microdomains (or lipid rafts). We have previously shown that alterations in cholesterol levels in myoblasts induce changes in proliferation and differentiation, which involves activation of Wnt/beta-catenin signaling pathway. In this study we used methyl-β-cyclodextrin (MbCD) to extract cholesterol from the membrane of chick skeletal muscle cells grown in culture. Using Ion Torrent-based sequencing, we compared the transcriptome of untreated and MbCD treated cells. Our aim was to define the genes that are expressed in these two conditions and relate their expression to cellular functions.ResultsOver 5.7 million sequences were obtained, representing 671.38 Mb of information. mRNA transcriptome profiling of myogenic cells after cholesterol depletion revealed alterations in transcripts involved in the regulation of apoptosis, focal adhesion, phagosome, tight junction, cell cycle, lysosome, adherens junctions, gap junctions, p53 signaling pathway, endocytosis, autophagy and actin cytoskeleton. Lim domain only protein 7 mRNA was found to be the highest up-regulated feature after cholesterol depletion.ConclusionsThis is the first study on the effects of membrane cholesterol depletion in mRNA expression in myogenic cells. Our data shows that alterations in the availability of plasma membrane cholesterol lead to transcriptional changes in myogenic cells. The knowledge of the genes involved in the cellular response to cholesterol depletion could contribute to our understanding of skeletal muscle differentiation.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-544) contains supplementary material, which is available to authorized users.

Transient cholesterol effects on nicotinic acetylcholine receptor cell-surface mobility.

To what extent do cholesterol-rich lipid platforms modulate the supramolecular organization of the nicotinic acetylcholine receptor (AChR)? To address this question, the dynamics of AChR particles at high density and its cholesterol dependence at the surface of mammalian cells were studied by combining total internal reflection fluorescence microscopy and single-particle tracking. AChR particles tagged with a monovalent ligand, fluorescent α-bungarotoxin (αBTX), exhibited two mobile pools: i) a highly mobile one undergoing simple Brownian motion (16%) and ii) one with restricted motion (∼50%), the rest being relatively immobile (∼44%). Depletion of membrane cholesterol by methyl-α-cyclodextrin increased the fraction of the first pool to 22% and 33% after 15 and 40 min, respectively; the pool undergoing restricted motion diminished from 50% to 44% and 37%, respectively. Monoclonal antibody binding results in AChR crosslinking-internalization after 2 h; here, antibody binding immobilized within minutes ∼20% of the totally mobile AChR. This proportion dramatically increased upon cholesterol depletion, especially during the initial 10 min (83.3%). Thus, antibody crosslinking and cholesterol depletion exhibited a mutually synergistic effect, increasing the average lifetime of cell-surface AChRs∼10 s to ∼20 s. The instantaneous (microscopic) diffusion coefficient D 2–4 of the AChR obtained from the MSD analysis diminished from ∼0.001 µm2 s−1 to ∼0.0001–0.00033 µm2 s−1 upon cholesterol depletion, ∼30% of all particles falling into the stationary mode. Thus, muscle-type AChR exhibits heterogeneous motional regimes at the cell surface, modulated by the combination of intrinsic (its supramolecular organization) and extrinsic (membrane cholesterol content) factors.

Localization of Kv4.2 and KChIP2 in lipid rafts and modulation of outward K+ currents by membrane cholesterol content in rat left ventricular myocytes.

Lipid rafts are cholesterol-enriched microdomains of the cell membrane. Here we investigate the localization of the pore forming K(+)-channel α-subunit Kv4.2 and the β-subunit KChIP2, underlying the transient outward K(+) current (I to), in lipid rafts in left ventricular myocytes. Furthermore, we explored the impact of membrane cholesterol depletion (using 20 mM methyl-beta-cyclodextrin (MBCD)) on K(+) outward currents. Cholesterol-saturated MBCD (20 mM) served as control. Myocytes were isolated from the left ventricular free wall of Wistar rats. The Triton X-100 (4 °C) insoluble fraction of whole cell protein was analyzed by sucrose density gradient centrifugation followed by Western blot. Kv4.2 and KChIP2 were partially detected in low-density fractions (lipid rafts). MBCD treatment (5 min) resulted in a shift of Kv4.2 and KChIP2 towards high-density fractions. K(+) currents were assessed by whole-cell patch-clamp. MBCD treatment resulted in a 29 ± 3 % decrease in I to (20.0 ± 1.6pApF(-1) vs. 28.5 ± 2.0pApF(-1), n = 15, p < 0.001, V Pip = 40 mV) within 5 min. Control solution resulted in a significantly smaller reduction in I to (17 ± 3 %, p < 0.001, p < 0.01 compared with MBCD). MBCD induced a 38 ± 9 % increase in the non-inactivating current component (I sus) (10.1 ± 0.6pApF(-1) vs. 7.6 ± 0.4pApF(-1), n = 15, p < 0.001). This effect was absent in control solution. The increase in I sus was not sensitive to 100 μM 4-aminopyridine or 20 mM tetraethylammonium, making a contribution of Kv1.5 or Kv2.1 unlikely. In conclusion, in rat ventricular cardiomyocytes, a fraction of Kv4.2 and KChIP2 is localized in lipid rafts. Membrane cholesterol depletion results in ~12 % net reduction of I to, a redistribution of the channel proteins Kv4.2 and KChIP2 and an increased delayed rectifier current.

Membrane cholesterol and caveolar modulation of cardiac function, ischemic tolerance and opioidergic protection (652.21)

Studies support a role for cholesterol‐rich membrane microdomains, caveolae, and caveolins in cardiac tolerance and conditioning to ischemia‐reperfusion (IR). The importance of membrane cholesterol in intrinsic IR tolerance and preconditioning is unclear. We assessed effects of cholesterol depletion vs. lifelong modulation of caveolin‐3 (deletion and overexpression) on myocardial function, IR tolerance and opioid preconditioning. Hearts from male C57/Bl6 mice received methyl‐β‐cyclodextrin (MβCD, 2 μM ‐ 1 mM), depleting membrane cholesterol, prior to ex vivo IR ± acute or sustained opioid receptor preconditioning (SLP). Additionally, cardiac caveolin‐3 (cav‐3) knockout (KO) and overexpressor (OE) hearts were exposed to IR ± SLP. MβCD significantly reduced sarcolemmal cholesterol content by &gt;10‐30%, and induced significant pre‐ and post‐ischemic contractile dysfunction. Cardioprotection with acute morphine (10 μM) was abolished with &gt;20 μM MβCD, yet SLP remained efficacious until 200 μM MβCD. Cav‐3 KO reduced, while cav‐3 OE enhanced IR tolerance. SLP improved post‐ischemic recovery in cav‐3 KO, and protection was additive in cav‐3 OE hearts. Membrane cholesterol plays a key role in intrinsic IR tolerance and conventional and novel preconditioning. Novel SLP is further delineated from conventional preconditioning stimuli, as this phenotype appears independent of cav‐3 protein.Grant Funding Source: Supported by National Heart Foundation Australia (LS) and Australian Research Council (JP)

Lipid Order Investigations Combined with Generalized Polarization Provide Deeper Insights into Plasma Membrane Architecture of Live Cells

The complexity of cell plasma membrane architecture is governed by biomolecular interactions, heterogeneity and complex sub-micrometric size domains. Even though there is no doubt about the existence of liquid-ordered and liquid-disordered phases in the model membranes, direct observations in live cells remain delicate due to their dynamic nature and sub-resolution size.High resolution optical microscopy techniques have helped the understanding of lipid organization and their contribution to biological functions. Among them, environmentally sensitive lipid probes (such as Laurdan or derivatives of ANEP) reveal lipid packing information (in particular fluidity governed by local polarity) at the nanometer scale, thanks to the bathochromic shift in their fluorescence emission spectrum as the membrane undergoes phase transition from gel to fluid [1]. The associated spectral ratiometric imaging is called generalized polarization (GP).In this work, we combine GP imaging with orientational investigations on such lipid probes, in order to probe, in a complementary approach, both local lipid packing (which reports a molecular scale information) and lipid order (which report a mesoscopic scale information). The orientational order of lipid probes, measured by fluorescence angle-resolved linear dichroism microscopy (FARLDM), has been previously shown to be highly sensitive to local membrane morphological changes (driven by cytoskeleton alterations) and cholesterol depletion [2].Implementing a combined GP-FARLDM technique, we show that new information can be gained on lipids interactions at different scales in the cell plasma membrane. We show in particular that liquid-ordered and liquid-disordered phases exhibit distinct morphological behaviors, and explore the capacities of different lipid probes to report information on the membrane architecture at the nanoscale.

Dextran-Based Doxorubicin Nanocarriers with Improved Tumor Penetration

Drug delivery systems with improved tumor penetration are valuable assets as anticancer agents. A dextran-based nanocarrier system with aldehyde functionalities capable of forming an acid labile linkage with the chemotherapy drug doxorubicin was developed. Aldehyde dextran nanocarriers (ald-dex-dox) demonstrated efficacy as delivery vehicles with an IC50 of ∼300 nM against two-dimensional (2D) SK-N-BE(2) monolayers. Confocal imaging showed that the ald-dex-dox nanocarriers were rapidly internalized by SK-N-BE(2) cells. Fluorescence lifetime imaging microscopy (FLIM) analysis indicated that ald-dex-dox particles were internalized as intact complexes with the majority of the doxorubicin released from the particle four hours post uptake. Accumulation of the ald-dex-dox particles was significantly enhanced by ∼30% in the absence of glucose indicating a role for glucose and its receptors in their endocytosis. However, inhibition of clathrin dependent and independent endocytosis and macropinocytosis as well as membrane cholesterol depletion had no effect on ald-dex-dox particle accumulation. In three-dimensional (3D) SK-N-BE(2) tumor spheroids, which more closely resemble a solid tumor, the ald-dex-dox nanoparticles showed a significant improvement in efficacy over free doxorubicin, as evidenced by decreased spheroid outgrowth. Drug penetration studies in 3D demonstrated the ability of the ald-dex-dox nanocarriers to fully penetrate into a SK-N-BE(2) tumor spheroids, while doxorubicin only penetrates to a maximum distance of 50 μM. The ald-dex-dox nanocarriers represent a promising therapeutic delivery system for the treatment of solid tumors due to their unique enhanced penetration ability combined with their improved efficacy over the parent drug in 3D.

Cholesterol lowering drug may influence cellular immune response by altering MHC II function

Major histocompatibility complex class II (MHC II) expressed on the surface of antigen-presenting cells (APCs) displays peptides to CD4⁺ T cells. Depletion of membrane cholesterol from APCs by methyl β-cyclodextrin treatment compromises peptide-MHC II complex formation coupled with impaired binding of conformational antibody, which binds close to the peptide binding groove of MHC II. Interestingly, the total cell surface of MHC II remains unaltered. These defects can be corrected by restoring membrane cholesterol. In silico docking studies with a three-dimensional model showed the presence of a cholesterol binding site in the transmembrane domain of MHC II (TM-MHC-II). From the binding studies it was clear that cholesterol, indeed, interacts with the TM-MHC-II and alters its conformation. Mutation of cholesterol binding residues (F240, L243, and F246) in the TM-MHC-II decreased the affinity for cholesterol. Furthermore, transfection of CHO cells with full-length mutant MHC II, but not wild-type MHC II, failed to activate antigen-specific T cells coupled with decreased binding of conformation-specific antibodies. Thus, cholesterol-induced conformational change of TM-MHC-II may allosterically modulate the peptide binding groove of MHC II leading to T cell activation.

Abstract 488: Atorvastatin Inhibits Src/ROS-mediated Redox Sensitive And Pro-Inflammatory Actions of Aldosterone in Vascular Smooth Muscle Cells: Focus on Statins Pleiotropic Effects

Statins, described as cholesterol-lowering drugs, are now recognized to induce effects throught pleiotropic actions, including oxidative stress reduction and protein geranylgeranylation inhibition. Mechanisms whereby aldosterone associates with c-Src and related redox signaling molecules involve lipid rafts. This study aims to identify whether c-Src/NADPH oxidases (Nox) pathway is a statin-sensitive target independent of membrane cholesterol depletion mechanisms. In order to discriminate statin pleiotropic effects from its classic cholesterol synthesis inhibition, VSMCs from WKY were treated with 1 uM atorvastatin for 60 min or 72h prior 100 nM aldosterone stimulations respectively. Aldosterone-induced c-Src phosphorylation (% vehicle, 267 ± 35) was inhibited by long (150 ± 23 %) and short (114 ± 33 %) term atorvastatin treatment. To restore cholesterol synthesis, cells were incubated with its intermediate, mevalonate (100 μM). Mevalonate reload restored c-Src phosphorylation (287 ± 27 %) induced by aldosterone in atorvastatin long term-treated VSMCs. Geranylgeranyl-pyrophosphate (GGPP) was used to prevent protein geranylgeranylation inhibition. GGPP reposition recovered the effects of aldosterone on c-Src phosphorylation in both short (268 ± 22 %) and long (203 ± 35 %) term VSMCs treated with atorvastatin. Aldosterone-induced increase of Nox1, 2 and 4 expression and the associated ROS-generation (215 ± 38 %) were inhibited by long and short term atorvastatin incubation. Aldosterone-induced RAC1/2 and p47phox translocation (cytosol to membrane) was prevented by atorvastatin treatments. Aldosterone stimulation increased Nox1 and p47phox content in cholesterol-enriched fractions, an effect inhibited by atorvastatin short term treatment. Atorvastatin also prevented the increase of redox-signaling phosphorylation (ERK1/2, p38 and JNK) and pro-inflammatory markers (VCAM-1 expression, NFkB p65 phosphorylation) by aldosterone. We demonstrate that atorvastatin influences c-Src/Nox-mediated effects of aldosterone involving lipid rafts through classic and pleiotropic actions independent of cholesterol depletion. This study identifies a novel mechanism for statins in aldosterone-associated vascular injury.

Protein τ-Mediated Effects on Rat Hippocampal Choline Transporters CHT1 and τ-Amyloid β Interactions

It is suggested that intracellular tau protein (τ), when released extracellularly upon neuron degeneration, could evoke direct toxic effects on the cholinergic neurotransmitter system through muscarinic receptors and thus contribute to the pathogenesis of Alzheimer's disease. In this study, we evaluated the in vitro effects of six naturally occurring monomeric τ isoforms on rat hippocampal synaptosomal choline transporters CHT1 (large transmembrane proteins associated with high-affinity choline transport and vulnerable to actions of amyloid β peptides (Aβ) applied in vitro or in vivo). Some τ isoforms at nM concentrations inhibited choline transport in a dose- and time-dependent saturable manner (352=441>410=383>381=412) and effects were associated with changes in the Michaelis constant rather than in maximal velocity. Moreover, the actions of τ 352/441 were not influenced by previous depolarisation of synaptosomes or by previous depletion of membrane cholesterol. Specific binding of [3H]hemicholinium-3 was not significantly altered by τ 352/441 at higher nM concentrations. Results of in vitro tests on CHT1 transporters from cholesterol-depleted synaptosomes supported interactions between Aβ 1-40 and τ 352. In addition, we developed surface plasmon resonance biosensors to monitor complexes of Aβ 1-42 and τ 352 using a sandwich detection format. It seems, therefore, that protein τ, similar to Aβ peptides, can contribute to the pathogenesis of Alzheimer's disease through its actions on CHT1 transporters. However, the interaction mechanisms are quite different (τ probably exerts its effects through direct interactions of microtubule binding repeats with extracellular portions of the CHT1 protein without influencing the choline recognition site, Aβ rather through lipid rafts in the surrounding membranes). An N-terminal insert of τ is not necessary but the N-terminal projection domain plays a role. The developed biosensor will be used to detect Aβ-τ complexes in cerebrospinal fluid in order to evaluate them as prospective biomarkers of Alzheimer's disease.

Clostridium difficile Binary Toxin CDT Induces Clustering of the Lipolysis-Stimulated Lipoprotein Receptor into Lipid Rafts

ABSTRACTClostridium difficile is the leading cause of antibiotics-associated diarrhea and pseudomembranous colitis. Hypervirulent C. difficile strains produce the binary actin-ADP-ribosylating toxin CDT (C. difficile transferase), in addition to the Rho-glucosylating toxins A and B. We recently identified the lipolysis-stimulated lipoprotein receptor (LSR) as the host receptor that mediates uptake of CDT into target cells. Here we investigated in H1-HeLa cells, which ectopically express LSR, the influence of CDT on the plasma membrane distribution of the receptor. We found by fluorescence microscopy that the binding component of CDT (CDTb) induces clustering of LSR into subcompartments of the plasma membrane. Detergent extraction of cells treated with CDTb, followed by sucrose gradient fractionation, uncovered accumulation of LSR in detergent-resistant membranes (DRMs) that contained typical marker proteins of lipid rafts. Membrane cholesterol depletion with methyl-β-cyclodextrin inhibited the association of LSR with DRMs upon addition of CDTb. The receptor-binding domain of CDTb also triggered LSR clustering into DRMs. CDTb-triggered clustering of LSR into DRMs could be confirmed in Caco-2 cells. Our data suggest that CDT forces its receptor to cluster into lipid rafts and that oligomerization of the B component might enhance but is not essential for this process.

Functional impact of cholesterol sequestration on actin cytoskeleton in normal and transformed fibroblasts

Membrane cholesterol and lipid rafts are implicated in various signalling processes involving actin rearrangement in living cells. However, functional link between raft integrity and organisation of cytoskeleton remains unclear. We have compared the effect of cholesterol sequestration on F-actin structures in normal and transformed fibroblasts in which microfilament system is developed to a different extent. The depletion of membrane cholesterol by methyl-beta-cyclodextrin (MbCD) resulted in a disruption of lipid rafts in plasma membrane as it was revealed by fluorescent labelling of GM1 ganglioside. In normal fibroblasts with highly developed microfilament system, the cholesterol depletion resulted in actin disassembly and reduction of stress fibres. However, in transformed cells containing low amount of fibrillar actin, MbCD treatment induced intensive formation of stress fibres and increased cell spreading. The results show that the effect of cholesterol depletion and lipid raft disruption on microfilament system is critically determined by the initial state of cytoskeleton, specifically, by the balance of polymerised and monomeric actin in the cell. We assume that uncapping of the microfilaments is the key step of cholesterol-regulated actin remodelling.

The novel endocannabinoid noladin ether holds putative anti-fibrotic properties by selectively inducing cell death in hepatic stellate cells

Background: We could previously demonstrate that the endogenous cannabinoid 2-arachidonoyl glycerol (2-AG) is an antifibrotic lipid mediator that blocks proliferation and induces apoptotic cell death in hepatic stellate cells (HSCs), but not in hepatocytes. Recently a novel endocannabinoid termed noladin ether (NE, 2-arachidonyl glyceryl ether) has been identified, which represents a more stable entity against enzymatic hydrolysis than 2-AG. Aims: To test the effects of NE on hepatic cell populations. Methods: Primary hepatocytes and HSCs were isolated from healthy rat or mouse liver by collagenase perfusion. Cell death was analyzed by LDH assay and western blot for caspase 3- and PARP-cleavage. Glutathione (GSH) was depleted by DL-buthionine-(S,R)-sulfoximine (BSO) treatment. Antioxidant resistance was enhanced by Trolox application. ROS formation was detected by DCFDA fluorescence. Membrane cholesterol was depleted by methyl-beta-cyclodextrin (MCD). Results: NE dose-dependently induced cell death in activated primary HSCs after 2–4h, starting from 2µM. In contrast to 2-AG, which induced late apoptosis in HSCs, NE induced hallmarks of necrosis, lack of caspase-3 or PARP cleavage. ROS formation in HSCs was significantly and even more increased by NE than by 2-AG. The antioxidant Trolox significantly decreased NE-induced death, thus indicating an ROS-dependent cell death mechanism. Although the cannabinoid receptors CB1, CB2 and TRPV1 were expressed in HSCs, pharmacological blockade failed to block NE-mediated cell death, indicating a cannabinoid-receptor independent mechanism. Interestingly, membrane cholesterol depletion with MCD completely inhibited NE-induced cell death. Similarly to 2-AG, NE did not induce cell death in hepatocytes. The resistance toward NE in hepatocytes was due to high levels of antioxidants such as GSH, since GSH depletion with BSO significantly increased NE-induced cell death in hepatocytes. Intriguingly, adenoviral overexpression of the putative endocannabinoid degradation enzyme fatty acid amide hydrolase (FAAH) in HSCs did not rescue from NE-induced cell death, underlining the stability of NE against enzymatic hydrolysis. Conclusion: NE effectively caused cell death in activated HSCs, but not in hepatocytes. NE-induced cell death was mediated through membrane cholesterol-rich lipid rafts and pronounced ROS formation, but independently from cannabinoid receptors. The selective induction of cell death in HSCs and its high stability against enzymatic hydrolysis proposes NE as a potential endogenous antifibrogenic mediator.

Membrane cholesterol modulates the hyaluronan-binding ability of CD44 in T lymphocytes and controls rolling under shear flow

The adhesion of circulating lymphocytes to the surface of vascular endothelial cells is important for their recruitment from blood to secondary lymphoid organs and to inflammatory sites. CD44 is a key adhesion molecule for this interaction and its ligand-binding ability is tightly regulated. Here we show that the hyaluronan-binding ability of CD44 in T cells is upregulated by the depletion of membrane cholesterol with methyl-β-cyclodextrin (MβCD), which disintegrates lipid rafts, i.e. cholesterol- and sphingolipid-enriched membrane microdomains. Increasing concentrations of MβCD led to a dose-dependent decrease in cellular cholesterol content and to upregulation of hyaluronan binding. Additionally, a cholesterol-binding agent filipin also increased hyaluronan binding. Cholesterol depletion caused CD44 to be dispersed from cholesterol-enriched membrane microdomains. Cholesterol depletion also increased the number of cells undergoing rolling adhesion under physiological flow conditions. Our results suggest that the ligand-binding ability of CD44 is governed by its cholesterol-dependent allocation to membrane microdomains at the cell surface. These findings provide novel insight into the regulation of T cell adhesion under blood flow.

Lipid Behavior in Integrin α2 Clustering

It is becoming increasingly more evident that transmembrane proteins can influence lipid organization in biological membranes. Our previous studies in model membranes have shown that peptides affect the partitioning of lipids in the bilayer. We now want to further investigate these protein-lipid interactions in a more biological context, by looking at integrin clustering in giant plasma membrane vesicles (GMPVs). Integrins are cell surface proteins that traverse the plasma membrane and mediate, for example, cell signaling and virus entry. Binding of Echovirus 1 to integrin α2 induces integrin clustering and the subsequent internalization of the virus-integrin complex. Clustering is crucial for virus entry, but can also be achieved through the addition of integrin specific antibodies to cells. We are interested in the possible reorganization of the membrane upon clustering as well as the influence of plasma membrane lipids on clustering. For this purpose we have prepared GMPVs from SAOS cells expressing α2 integrin, and induced antibody-mediated clustering in them. We are using lipid probes to visualize the liquid ordered and liquid disordered phase, and fluorescent secondary antibodies to detect α2 integrin in the GPMVs with confocal microscopy. We will look at lipid organization in cells expressing integrin mutants, which are unable to cluster and/or internalize. And we will treat the cells and/or GPMVs in ways that alter their lipid content, such as deplete membrane cholesterol with cyclodextrin, and monitor the resulting changes in integrin and lipid behavior in GMPVs. With these experiments we hope to gain further insight into the interplay of transmembrane proteins and lipids in biological membranes. Also, understanding the lipid behavior in integrin clustering will aid in understanding what governs clustering and how it affects membrane architecture. This information can in turn be beneficial for developing ways of preventing virus entry.

Regulation of CAMP Compartmentation by Membrane Microdomains

The role of different membrane domains in the compartmentation of cAMP signaling was investigated using the FRET-based biosensor Epac2-camps. The MyrPalm sequence from Lyn kinase and the CAAX sequence from Rho GTPase were used to target this probe to lipid raft (Epac2-MyrPalm) or non-lipid raft (Epac2-CAAX) domains, respectively. Confocal imaging established that both probes were targeted to the plasma membrane in HEK293 cells. FRAP analysis demonstrated that depletion of membrane cholesterol altered both the recovery half-time and the mobile fraction of Epac2-MyrPalm but not Epac2-CAAX, confirming that each probe was targeted to the correct microdomain. FRET responses of these probes were then used to monitor relative changes in cAMP activity associated with lipid raft and non-raft domains. The results demonstrated that basal cAMP activity is significantly higher in non-raft domains. This was supported by the fact that the maximal increase in cAMP over baseline following agonist stimulation was significantly smaller for Epac2-CAAX than it was for Epac2-MyrPalm, consistent with the idea that the probe was partially activated by a higher basal level of cAMP associated with non-lipid raft domains. In addition, inhibition of adenylyl cyclase activity with MDL 12330A reduced basal cAMP activity detected by Epac2-CAAX but not Epac2-MyrPalm. Responses detected by Epac2-CAAX were also more sensitive to direct activation of adenylyl cyclase by forskolin, but less sensitive to inhibition of type 4 phosphodiesterase activity by rolipram. These results indicate that there are diffusionally-restricted pools of cAMP associated with different membrane microdomains under basal conditions. The higher basal cAMP activity associated with non-lipid raft domains can be explained by differences in basal adenylyl cyclase and phosphodiesterase activity.

DNA uptake in swine sperm: Effect of plasmid topology and methyl‐beta‐cyclodextrin‐mediated cholesterol depletion

Sperm-mediated gene transfer (SMGT), the ability of sperm cells to spontaneously incorporate exogenous DNA and to deliver it to oocytes during fertilization, has been proposed as an easy and efficient method for producing transgenic animals. SMGT is still undergoing development and optimization to improve the uptake efficiency of foreign DNA by sperm cells, which is a preliminary, yet critical, step for successful SMGT. Towards this aim, we developed a quantitative, real-time PCR-based assay to assess the absolute number of exogenous plasmids internalized into the spermatozoon. Using this technique, we found that the circular form of the DNA is more efficiently taken up than the linearized form. We also found that DNA internalization into the nucleus of porcine sperm cells is better under specific methyl-β-cyclodextrin (MCD)-treated conditions, where the plasma membrane properties were altered without significantly compromising sperm physiology. These results provide the first evidence that membrane cholesterol depletion by MCD might represent a novel strategy for enhancing the ability of sperm to take up heterologous DNA.

Membrane Cholesterol Strongly Influences Confined Diffusion of Prestin

Prestin is the membrane motor protein that drives outer hair cell (OHC) electromotility, a process that is essential for mammalian hearing. Prestin function is sensitive to membrane cholesterol levels, and numerous studies have suggested that prestin localizes in cholesterol-rich membrane microdomains. Previously, fluorescence recovery after photobleaching experiments were performed in HEK cells expressing prestin-GFP after cholesterol manipulations, and revealed evidence of transient confinement. To further characterize this apparent confined diffusion of prestin, we conjugated prestin to a photostable fluorophore (tetramethylrhodamine) and performed single-molecule fluorescence microscopy. Using single-particle tracking, we determined the microscopic diffusion coefficient from the full time course of the mean-squared deviation. Our results indicate that prestin undergoes diffusion in confinement regions, and that depletion of membrane cholesterol increases confinement size and decreases confinement strength. By interpreting the data in terms of a mathematical model of hop-diffusion, we quantified these cholesterol-induced changes in membrane organization. A complementary analysis of the distribution of squared displacements confirmed that cholesterol depletion reduces prestin confinement. These findings support the hypothesis that prestin function is intimately linked to membrane organization, and further promote a regulatory role for cholesterol in OHC and auditory function.

Porphyromonas gingivalis entry into gingival epithelial cells modulated by Fusobacterium nucleatum is dependent on lipid rafts

Host cell invasion by a major periodontal pathogen, Porphyromonas gingivalis, has been proposed as an important mechanism involved in host–pathogen interactions in periodontal and cardiovascular diseases. The present study sought to gain insight into the underlying mechanism(s) involved in previously demonstrated fusobacterial modulation of host cell invasion by P. gingivalis. An immortalized human gingival cell line Ca9-22 was dually infected with P. gingivalis ATCC 33277 and Fusobacterium nucleatum TDC 100, and intracellular invasion was assessed by scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). SEM observation showed that P. gingivalis and F. nucleatum formed consortia and were in the process of penetrating into Ca9-22 by 30–60 min after infection. In CSLM, Ca9-22 cells that contained both P. gingivalis and F. nucleatum were frequently observed after 2 h, although cells that contained exclusively P. gingivalis were also found. Infection by P. gingivalis and/or F. nucleatum revealed evident colocalization with a lipid raft marker, GM1-containing membrane microdomains. In an antibiotic protection assay, depletion of epithelial plasma membrane cholesterol resulted in a significant reduction of recovered P. gingivalis or F. nucleatum (∼33% of untreated control; p < 0.001). This inhibition was also confirmed by CSLM. Sequential infection experiments showed that timing of infection by each species could critically influence the invasion profile. Co-infection with F. nucleatum significantly enhanced host cell invasion by P. gingivalis 33277, its serine phophatase SerB mutant and complemented strains, suggesting that the SerB does not play a major role in this fusobacterial enhancement of P. gingivalis invasion. Thus, the interaction between F. nucleatum and host cells may be important in the fusobacterial enhancement of P. gingivalis invasion. Collectively, these results suggest that lipid raft-mediated process is at least one of the potential mechanisms involved in fusobacterium-modulated host cell invasion by P. gingivalis.