Cholesterol-rich Rafts Research Articles

Cholesterol-Depletion-Induced Membrane Repair Carries a Raft Conformer of P-Glycoprotein to the Cell Surface, Indicating Enhanced Cholesterol Trafficking in MDR Cells, Which Makes Them Resistant to Cholesterol Modifications.

The human P-glycoprotein (P-gp), a transporter responsible for multidrug resistance, is present in the plasma membrane's raft and non-raft domains. One specific conformation of P-gp that binds to the monoclonal antibody UIC2 is primarily associated with raft domains and displays heightened internalization in cells overexpressing P-gp, such as in NIH-3T3 MDR1 cells. Our primary objective was to investigate whether the trafficking of this particular P-gp conformer is dependent on cholesterol levels. Surprisingly, depleting cholesterol using cyclodextrin resulted in an unexpected increase in the proportion of raft-associated P-gp within the cell membrane, as determined by UIC2-reactive P-gp. This increase appears to be a compensatory response to cholesterol loss from the plasma membrane, whereby cholesterol-rich raft micro-domains are delivered to the cell surface through an augmented exocytosis process. Furthermore, this exocytotic event is found to be part of a complex trafficking mechanism involving lysosomal exocytosis, which contributes to membrane repair after cholesterol reduction induced by cyclodextrin treatment. Notably, cells overexpressing P-gp demonstrated higher total cellular cholesterol levels, an increased abundance of stable lysosomes, and more effective membrane repair following cholesterol modifications. These modifications encompassed exocytotic events that involved the transport of P-gp-carrying rafts. Importantly, the enhanced membrane repair capability resulted in a durable phenotype for MDR1 expressing cells, as evidenced by significantly improved viabilities of multidrug-resistant Pgp-overexpressing immortal NIH-3T3 MDR1 and MDCK-MDR1 cells compared to their parents when subjected to cholesterol alterations.

Raft platforms highly enriched in cholesterol: major scaffolds for IL-6 signalling assembly with implications in inflammation and cancer.

Interleukin-6 (IL-6) is a pleiotropic cytokine with complex and major roles in inflammation, which could be linked to the different ways IL-6 signals at the plasma membrane. In this issue of FEBS Journal, Woo and co-workers present evidence for the involvement of Eps15 homology domain-containing protein 1 (EHD1)-mediated lipid raft platforms, highly enriched in cholesterol, in the IL-6 signalling pathway. Because of the strong connection between IL-6, inflammation and cancer, one implication of this report is that agents or approaches targeting cholesterol-rich raft platforms may assist the development of novel strategies to treat inflammatory and malignant diseases. Comment on: https://doi.org/10.1111/febs.16458.

Interplay between Caveolin-1 and body and tumor size affects clinical outcomes in breast cancer

BackgroundCaveolin-1 (CAV1) is associated with cholesterol-rich membrane raft domains and is a master regulator of cell signaling and membrane transport. Here, we investigated CAV1’s role in cellular compartments of breast cancer in relation to signaling pathways, clinicopathological features, and clinical outcomes. MethodsCAV1 levels were evaluated with immunohistochemistry in cytoplasm of invasive tumor cells and stromal cells in tumor tissue microarrays from a cohort of 1018 breast cancer patients (inclusion 2002–2012, Sweden). Cytoplasmic and stromal CAV1 were categorized as positive/negative and strong/not strong, respectively. CAV1 expression in relation to clinical outcomes was assessed with Cox regression. Investigations into CAV1 functional pathways was conducted in the STRING, GOBO, and TCGA databases. ResultsCAV1 expression was associated with non-luminal subtypes, cell cycle control, inflammation, epithelial-mesenchymal transition, and the IGF/Insulin system. Generally, CAV1 was not associated with recurrence risk. Stromal CAV1’s impact on recurrence risk was modified by BMI ≥25 kg/m2 (Pinteraction = 0.002), waist ≥80 cm (Pinteraction = 0.005), and invasive tumor size (pT2/3/4) (Pinteraction = 0.028). In low-risk patients only, strong stromal CAV1 significantly increased recurrence risk (HRsadj ≥1.61). In all patients, positive cytoplasmic CAV1 conferred >2-fold risk for contralateral disease HRadj 2.63 (95% CI 1.36–5.10). Strong stromal CAV1 conferred nearly 2-fold risk for locoregional recurrence HRadj 1.88 (95% CI 1.09–3.24). ConclusionsCAV1’s prognostic impact depended on its localization, anthropometric, and tumor factors. Stromal CAV1 predicted high recurrence risk in a group of supposedly ‘low-risk’ patients. Cytoplasmic CAV1 predicted metachronous contralateral disease. If confirmed, CAV1 could be used as treatment target and for risk-stratification.

Cholesterol-Rich Lipid Rafts in the Cellular Membrane Play an Essential Role in Avian Reovirus Replication.

Cholesterol is an essential component of lipid rafts in cellular plasma membranes. Although lipid rafts have been reported to have several functions in multiple stages of the life cycles of many different enveloped viruses, the mechanisms by which non-enveloped viruses, which lack outer lipid membranes, infect host cells remain unclear. In this study, to investigate the dependence of non-enveloped avian reovirus (ARV) infection on the integrity of cholesterol-rich membrane rafts, methyl-β-cyclodextrin (MβCD) was used to deplete cellular membrane cholesterol at the ARV attachment, entry, and post-entry stages. Treatment with MβCD significantly inhibited ARV replication at both the entry and post-entry stages in a dose-dependent manner, but MβCD had a statistically insignificant effect when it was added at the attachment stage. Moreover, MβCD treatment markedly reduced syncytium formation, which occurs at a relatively late stage of the ARV life cycle and is involved in cell-cell transmission and release. Furthermore, the addition of exogenous cholesterol reversed the effects mentioned above. Colocalization data also showed that the ARV proteins σC, μNS, and p10 prefer to localize to cholesterol-rich lipid raft regions during ARV infection. Altogether, these results suggest that cellular cholesterol in lipid rafts plays a critical role in ARV replication.

Function of Platelet Glycosphingolipid Microdomains/Lipid Rafts.

Lipid rafts are dynamic assemblies of glycosphingolipids, sphingomyelin, cholesterol, and specific proteins which are stabilized into platforms involved in the regulation of vital cellular processes. The rafts at the cell surface play important functions in signal transduction. Recent reports have demonstrated that lipid rafts are spatially and compositionally heterogeneous in the single-cell membrane. In this review, we summarize our recent data on living platelets using two specific probes of raft components: lysenin as a probe of sphingomyelin-rich rafts and BCθ as a probe of cholesterol-rich rafts. Sphingomyelin-rich rafts that are spatially and functionally distinct from the cholesterol-rich rafts were found at spreading platelets. Fibrin is translocated to sphingomyelin-rich rafts and platelet sphingomyelin-rich rafts act as platforms where extracellular fibrin and intracellular actomyosin join to promote clot retraction. On the other hand, the collagen receptor glycoprotein VI is known to be translocated to cholesterol-rich rafts during platelet adhesion to collagen. Furthermore, the functional roles of platelet glycosphingolipids and platelet raft-binding proteins including G protein-coupled receptors, stomatin, prohibitin, flotillin, and HflK/C-domain protein family, tetraspanin family, and calcium channels are discussed.

U18666A inhibits classical swine fever virus replication through interference with intracellular cholesterol trafficking

The level of cholesterol in host cells has been demonstrated to affect viral infection. Our previous studies showed that cholesterol-rich membrane rafts mediated the entry of classical swine fever virus (CSFV) into PK-15 or 3D4/21 cells, but the role of cholesterol post entry was still not clear. In this study, we found that CSFV replication before fusion was affected when the cholesterol trafficking in infected cells was disrupted using a cholesterol transport inhibitor, U18666A. Our data showed that U18666A affected both the fusion and replication steps in the life cycle of the virus, but not its binding and entry steps. The subsequent experiments confirmed that niemann-pick C1 (NPC1), a lysosomal membrane protein that helps cholesterol to leave the lysosome, was affected by U18666A, which led to the accumulation of cholesterol in lysosomes and inhibition of CSFV replication. Imipramine, a cationic hydrophobic amine similar to U18666A, also inhibited CSFV replication via similar mechanism. Surprisingly, the antiviral effect of U18666A was restored by the histone deacetylase inhibitor (HDACi), Vorinostat, which suggested that HDACi reverted the dysfunction of NPC1, and intra-cellular cholesterol accumulation disappeared and CSFV replicability resumed. Together, these data indicated that CSFV transformed from early endosome and late endosome into lysosome after endocytosis for further replication and that U18666A was a potential drug candidate for anti-pestivirus treatment.

Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane.

Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1.IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships between viral proteins in the plasma membrane. Some proteins, such as HA and M2, inherently cocluster within the membrane, although M2 is found mostly at the periphery of regions of HA, consistent with the proposed role of M2 in scission at the end of budding. The association between some pairs of influenza virus proteins, such as M2 and NP, appears to be brokered by additional influenza virus proteins, in this case M1. HA and NA, while raft associated, reside in distinct domains, reflecting their distributions in the viral membrane.

Molecular Dynamics Simulations of Amyloid β-Peptide (1-42): Tetramer Formation and Membrane Interactions

The aggregation cascade and peptide-membrane interactions of the amyloid β-peptide (Aβ) have been implicated as toxic events in the development and progression of Alzheimer’s disease. Aβ42 forms oligomers and ultimately plaques, and it has been hypothesized that these oligomeric species are the main toxic species contributing to neuronal cell death. To better understand oligomerization events and subsequent oligomer-membrane interactions of Aβ42, we performed atomistic molecular-dynamics (MD) simulations to characterize both interpeptide interactions and perturbation of model membranes by the peptides. MD simulations were utilized to first show the formation of a tetramer unit by four separate Aβ42 peptides. Aβ42 tetramers adopted an oblate ellipsoid shape and showed a significant increase in β-strand formation in the final tetramer unit relative to the monomers, indicative of on-pathway events for fibril formation. The Aβ42 tetramer unit that formed in the initial simulations was used in subsequent MD simulations in the presence of a pure POPC or cholesterol-rich raft model membrane. Tetramer-membrane simulations resulted in elongation of the tetramer in the presence of both model membranes, with tetramer-raft interactions giving rise to the rearrangement of key hydrophobic regions in the tetramer and the formation of a more rod-like structure indicative of a fibril-seeding aggregate. Membrane perturbation by the tetramer was manifested in the form of more ordered, rigid membranes, with the pure POPC being affected to a greater extent than the raft membrane. These results provide critical atomistic insight into the aggregation pathway of Aβ42 and a putative toxic mechanism in the pathogenesis of Alzheimer’s disease.

Biological relevance of fatty acyl heterogeneity to the neural membrane dynamics of rhesus macaques during normative aging.

Lipidomic analyses of the frontal cortex of Rhesus macaques across three selected age groups (young, sexually-mature, old) revealed that docosahexaenoic acids (DHAs) displayed notable and unique accretions in sexually-mature macaques for all phospholipid classes examined, which were not observable in all remaining polyunsaturated fatty acids (PUFAs) investigated. On the other hand, arachidonic acid (ARA) exhibited sharp attritions in the membrane lipidomes of sexually-mature macaques, a decline which was attenuated only for cardiolipins (CLs). DHA enrichment in phospholipids was lost in old macaques, with accompanying augmentations in very-long-chain sphingomyelins (VLC-SMs). Age-dependent alterations in membrane lipidomes point to a possibly complex temporal interplay between DHA-enriched membrane microdomains and SM-/cholesterol-rich rafts in neural membranes during normative aging. Lipid co-regulation data revealed an increasingly intense degree of co-regulation between membrane lipid classes with age, and suggest that reduction in CLs during normative brain aging may prompt alternative membrane lipid synthetic pathways driven by a compromised energy availability in the aging brain.

Rac1 Participates in Thermally Induced Alterations of the Cytoskeleton, Cell Morphology and Lipid Rafts, and Regulates the Expression of Heat Shock Proteins in B16F10 Melanoma Cells

Eukaryotic cells exhibit a characteristic response to hyperthermic treatment, involving morphological and cytoskeletal alterations and the induction of heat shock protein synthesis. Small GTPases of the Ras superfamily are known to serve as molecular switches which mediate responses to extracellular stimuli. We addressed here how small GTPase Rac1 integrates signals from heat stress and simultaneously induces various cellular changes in mammalian cells. As evidence that Rac1 is implicated in the heat shock response, we first demonstrated that both mild (41.5°C) and severe (43°C) heat shock induced membrane translocation of Rac1. Following inhibition of the activation or palmitoylation of Rac1, the size of its plasma membrane-bound pool was significantly decreased while the heat shock-induced alterations in the cytoskeleton and cell morphology were prevented. We earlier documented that the size distribution pattern of cholesterol-rich rafts is temperature dependent and hypothesized that this is coupled to the triggering mechanism of stress sensing and signaling. Interestingly, when plasma membrane localization of Rac1 was inhibited, a different and temperature independent average domain size was detected. In addition, inhibition of the activation or palmitoylation of Rac1 resulted in a strongly decreased expression of the genes of major heat shock proteins hsp25 and hsp70 under both mild and severe heat stress conditions.

Regulation of the high‐affinity choline transporter activity and trafficking by its association with cholesterol‐rich lipid rafts

The sodium-coupled, hemicholinium-3-sensitive, high-affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts in both SH-SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH-SY5Y cells expressing rat CHT with filipin, methyl-β-cyclodextrin (MβC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol-saturated MβC. Kinetic analysis of binding of [(3)H]hemicholinium-3 to CHT revealed that reducing membrane cholesterol with MβC decreased both the apparent binding affinity (KD) and maximum number of binding sites (Bmax ); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol-rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis. The sodium-coupled choline transporter CHT moves choline into cholinergic nerve terminals to serve as substrate for acetylcholine synthesis. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts, and decreasing membrane cholesterol significantly reduces both choline uptake activity and cell surface CHT protein levels. CHT association with cholesterol-rich rafts is critical for its function, and alterations in plasma membrane cholesterol could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis.

Statins cause profound effects on gene expression in human cancer cells in vitro: the role of membrane microdomains.

There is increasing evidence that statin treatment can be beneficial in certain cancer patients. To determine if these benefits are a direct result of the cholesterol-lowering effects of statins or a result of secondary, protein transcription effects, the impacts of pravastatin and a cholesterol sequestrating agent methyl-beta-cyclodextrin (MbetaCD) on mRNA expression in the breast cancer cell MDA-MB-231 and the lung carcinoma cell Calu-1 have been compared by microarray techniques. The effects of these agents on cholesterol-rich rafts and caveolae, which have significance in cancer signaling, have also been examined. Both treatments caused a general downregulation of not only signal transduction including cancer pathway proteins, but also apoptosis and chemokine pathways, with statins impacting 35 genes by twofold or greater in MDA-MB-231 and > 300 genes in Calu-1. These manifold dysregulations could also explain the various side effects reportedly caused by statins. MbetaCD produced far fewer statistical events than pravastatin in the breast cancer line but many more in the lung cell line. Pravastatin increased expression of CAV1 but caveolae density decreased and overall raft density was unaffected. MbetaCD also caused an increase in CAV1 expression and reduced the prevalence of both rafts and caveolae. It is proposed that sequestration of cholesterol from the membrane by MbetaCD is not equivalent to blockade of the cholesterol pathway and causes different effects on microdomain-mediated signal transduction dependant on the cell line. The profound effects of statins on mRNA expression can be explained by the failure of caveolin-1 to properly complex with cholesterol in an altered sterol environment, with caveolae acting as the main loci for signaling directed towards those transcription processes unaffected by MbetaCD. Targeted inhibition of the postmevalonate pathway could offer an opportunity to specifically reduce caveolae-based signaling in cancer cells. The observed impact of pravastatin on gene expression may explain the pleiotropic effects of statins when they are used as adjuvants in chemotherapy and suggests impact on gene expression as a possible cause of side effects from statin use.

Abstract 4287: Intracellular trafficking of androgen receptor splice variant AR8.

Abstract In a classic paradigm, the intracellular trafficking of a ligand-stimulated androgen receptor involves receptor dimerization, dissociating from chaperones (such as HSPs), interact with nuclear membrane importin and Ran-GTPase to gain access to nucleus; once there, it complexes with transcriptional regulators and binding to target sites. Despite the compelling evidence for AR non-genomic signaling events, the molecular mechanism of how does AR signaling from cytoplasmic or membrane remains undetermined. Several recent studies suggested that membrane association of AR is mediated by palmitoylation of AR at cysteine-806. Two plamitoylation enzymes, DHHC7 and DHHC21, have been implicated in the process of AR palmitoylation. In the current study, we determined the intracellular trafficking of a newly identified AR isoform, AR8. AR8 is a splice variant of the original full-length AR. AR8 was demonstrated to be up-regulated in castration-resistant prostate cancer cells. Structurally, AR8 protein sequence is different from other AR variants with a substitution of all the amino acids after N-terminal domain (NTD) of a conventional AR with an unique C-terminal sequence. Coincidentally, two cysteine residues within the unique c-terminus, positions 558 and 560, were identified to be palmitoylated. The palmitoylation was determined to be responsible for the localization of AR8 to plasma membrane. Using a fluorescent protein mCherry-tagged AR8 and confocal microscopy, we confirmed the plasma membrane localization of AR8 as previously described. We determined that AR8 co-localized with a Golgi marker GM-130 at peri-nuclear regions by immunofluorescence microscopy. The result was further substantiated by co-localizing mCherry-AR8 with galactosyltransferase (GalT) golgi-targeting sequence-fused GFP (GalT-GFP; green fluorescent protein) at Golgi compartments. We also demonstrated that mCherry-AR8 co-localized with EGFR-GFP (epidermal growth factor receptor-GFP) on the plasma membrane within in the cholesterol rich raft domain. Using time-lapse confocal microscopy, we observed that AR8 co-migrated with EGFR in a retrograde fashion and become associated with endosomal compartments at the onset of EGF (5ng/ml) stimulation. Part of these endosomes was later fused with lysosomes as evident from co-localizations of mCherry-AR8 with GFP-LAMP-1. Although the significance of AR8 expression in the progression of prostate cancer remains to be determined, our observations underline a potential intracellular trafficking pathway associated with AR8 are consistent with the original report that EGF stimulation promotes the interactions between AR8 and EGFR. Citation Format: Ciny John, Michael L. Lu. Intracellular trafficking of androgen receptor splice variant AR8. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4287. doi:10.1158/1538-7445.AM2013-4287

Ezrin/Radixin/Moesin Proteins and Flotillins Cooperate to Promote Uropod Formation in T Cells

T cell uropods are enriched in specific proteins including adhesion receptors such as P-selectin glycoprotein ligand-1 (PSGL-1), lipid raft-associated proteins such as flotillins and ezrin/radixin/moesin (ERM) proteins which associate with cholesterol-rich raft domains and anchor adhesion receptors to the actin cytoskeleton. Using dominant mutants and siRNA technology we have tested the interactions among these proteins and their role in shaping the T cell uropod. Expression of wild type (WT) ezrin-EGFP failed to affect the morphology of human T cells or chemokine-induced uropod recruitment of PSGL-1 and flotillin-1 and -2. In contrast, expression of constitutively active T567D ezrin-EGFP induced a motile, polarized phenotype in some of the transfected T cells, even in the absence of chemokine. These cells featured F-actin-rich ruffles in the front and uropod enrichment of PSGL-1 and flotillins. T567D ezrin-EGFP was itself strongly enriched in the rear of the polarized T cells. Uropod formation induced by T567D ezrin-EGFP was actin-dependent as it was attenuated by inhibition of Rho-kinase or myosin II, and abolished by disruption of actin filaments. While expression of constitutively active ezrin enhanced cell polarity, expression of a dominant-negative deletion mutant of ezrin, 1–310 ezrin-EGFP, markedly reduced uropod formation induced by the chemokine SDF-1, T cell front-tail polarity, and capping of PSGL-1 and flotillins. Transfection of T cells with WT or T567D ezrin did not affect chemokine-mediated chemotaxis whereas 1–310 ezrin significantly impaired spontaneous 2D migration and chemotaxis. siRNA-mediated downregulation of flotillins in murine T cells attenuated moesin capping and uropod formation, indicating that ERM proteins and flotillins cooperate in uropod formation. In summary, our results indicate that activated ERM proteins function together with flotillins to promote efficient chemotaxis of T cells by structuring the uropod of migrating T cells.

Reversible clustering of sodium channels at the spike is driven by a local crystallization/melting transition around bilayer rafts

The classical theory of nerve signal conduction, introduced half a century ago by Hodgkin and Huxley, was challenged in 2005 by the so called soliton theory introduced by Heimburg and Jackson. This latter theory is based on a bilayer phase transition forming a mechanical wave, involving a transition from a disordered conformation of the bilayer into the gel-state, and the electric pulse is regarded a result of the phase transition. We support the phase transition concept, and provide evidence for a reversible clustering process at the spike. This clustering involves cholesterol-rich rafts, where the voltage-gated sodium channels are located of the sodium channels at the spike region. The clustering is driven by local crystallization into the gel-type of bilayer conformation around the rafts, and is explained by the hexagonal chain organization in the gel-phase related to that in the raft structure. The clustering is reversed at the melting transition, and the role of the action potential in these phase transition is finally considered.

Lipid Rafts and Fas/CD95 Signaling in Cancer Chemotherapy

Cholesterol- and sphingolipid-rich membrane domains, termed lipid rafts, have been recently involved in the triggering of death receptor-mediated apoptosis. The alkyl-lysophospholipid analogue edelfosine was the first antitumor drug reported to induce apoptosis in cancer cells through co-clustering of lipid rafts and Fas/CD95 death receptor. Recruitment and aggregation of Fas/CD95 in lipid raft clusters was independent of its cognate ligand FasL/CD95L, and could be pharmacologically modulated. The adaptor molecule Fas-associated death domain protein (FADD) and procaspase-8 were also recruited into lipid rafts following edelfosine treatment, forming the death-inducing signaling complex (DISC), and hence these membrane microdomains can act as scaffolds for Fas/CD95 death signaling. Edelfosine accumulated in lipid rafts of cancer cells, altering raft protein and lipid composition. Subsequently, an increasing number of antitumor drugs have been found to induce apoptosis through recruitment of Fas/CD95 into membrane rafts, and some of these compounds accumulated in raft membrane domains. Additional downstream apoptotic signaling molecules have also been reported to be recruited into rafts following treatment of cancer cells with antitumor agents, thus facilitating protein-protein interactions and conveying apoptotic signals. On these grounds, lipid rafts have become an appealing and promising target for therapeutic intervention in cancer chemotherapy. Co-clustering of lipid rafts and Fas/CD95 signaling provides a new insight in the regulation of death receptor-mediated apoptosis, opening a new avenue in cancer therapy. In this regard, an increasing number of patents are dealing with the above insights in order to improve cancer treatment.

Role of Muscle Specific Caveolin-3 in Mechanosensitive Channel Regulation

Caveolin-3 (Cav-3) is a small muscle specific scaffolding protein that acts to complex signaling proteins within cholesterol rich raft domains through a small cytoplasmic binding domain. Cav-3 has the unique function of inserting only into the inner membrane leaflet causing expansion of the inner surface that induces curvature and membrane involutions called caveolae. In muscle the dystroglycan complex is localized to the caveolae and is the cortical cytoskeleton attachment point for dystrophin and other proteins which regulate sarcolemma tension. These attributes make caveolae ideal mechanosensing structures and likely locations for mechanosensitive channels (MSCs). To understand the role of Cav-3 in MSC regualtion we depleted membrane cholesterol with methyl β-cyclodextran which caused disruption and internalization of the caveolae protein and increased MSC activity in patch pipette recordings. Overexpression of a Cav-3-GFP fusion protein in well differentiated myotubes also leads to increased MSC activity. Reduction of Cav-3 with miRNA produced no change in MSC activity, though baseline MSC activity is already low in differentiated myotubes. To rapidly and specifically inhibit Cav-3 interactions, we fused the 20 amino acid Cav-3 scaffolding domain the 16 amino acid antennapedia membrane translocation signaling domain (Anten-CSD). Treatment of myotubes with 20 μM Anten-CSD caused internal Ca2+ to increase, large blebs to formed on the sarcolemma and Cav-3-GFP aggregation on the surface. There was an increase in MSC activity within 20 minutes after Anten-CBD treatment. We are currently investigating a link between Cav-3 and TRP channels. These studies suggest that Cav-3 may be involved in both localization of MSCs to the sarcolemma and regulating channel activity in myotubes through its ability to complex MSCs with the cortical cytoskeleton.

Impact of oxLDL on Cholesterol-Rich Membrane Rafts

Numerous studies have demonstrated that cholesterol-rich membrane rafts play critical roles in multiple cellular functions. However, the impact of the lipoproteins on the structure, integrity and cholesterol composition of these domains is not well understood. This paper focuses on oxidized low-density lipoproteins (oxLDLs) that are strongly implicated in the development of the cardiovascular disease and whose impact on membrane cholesterol and on membrane rafts has been highly controversial. More specifically, we discuss three major criteria for the impact of oxLDL on membrane rafts: distribution of different membrane raft markers, changes in membrane cholesterol composition, and changes in lipid packing of different membrane domains. We also propose a model to reconcile the controversy regarding the relationship between oxLDL, membrane cholesterol, and the integrity of cholesterol-rich membrane domains.

α V β 3 -integrin routes herpes simplex virus to an entry pathway dependent on cholesterol-rich lipid rafts and dynamin2

HSVs enter cells in a receptor-dependent [nectin1 or herpesviruses entry mediator (HVEM)] fashion by fusion of the viral envelope with plasma membrane (neutral pH compartment), by endocytosis into neutral or acidic compartments, or by macropinocytosis/phagocytosis. The cellular determinants of the route of entry are unknown. Here, we asked what cellular factors determine the pathway of HSV entry. CHO cells lack β(3)-integrin and the respective α-subunits' heterodimers. We report that, in the absence of α(V)β(3)-integrin, HSV enters CHO-nectin1 cells through a pathway independent of cholesterol-rich rafts and dynamin2. In the presence of α(V)β(3)-integrin, HSV enters CHO-nectin1 cells through a pathway dependent on cholesterol-rich rafts and dynamin2. HSV enters J-nectin1 and 293T cells through a neutral compartment independent of cholesterol-rich rafts and dynamin2. α(V)β(3)-integrin overexpression in these cells modifies the route of entry to an acidic compartment dependent on cholesterol-rich rafts and dynamin2, hence similar to that in α(V)β(3)-integrin-positive CHO-nectin1 cells. In some cells, the diversion of entry from an integrin- and raft-independent pathway to an acidic compartment requiring cholesterol-rich lipids rafts and dynamin2 is irreversible. Indeed, HSV cannot infect CHO-nectin1-α(V)β(3) cells through any compartment when the αvβ3-integrin-dependent pathway is blocked by anti-integrin antibody, anti-dynamin2, or anti-acidification drugs. We conclude that the αvβ3-integrin is a determinant in the choice of HSV entry pathway into cells. Because the pathway dictated by αvβ3-integrin is through lipid rafts, the platforms for a number of Toll-like receptors, current findings raise the possibility that αvβ3-integrin acts as a sentinel of innate immunity.

Intra-Clonal Diversity Identified by CD19-Mediated Signaling Correlates with Disease Progression In Chronic Lymphocytic Leukemia (CLL)

AbstractAbstract 3577Emerging data on intra-clonal diversity imply that this phenomenon may play a role in the clinical outcome of patients with CLL. Subsets of the CLL clone that respond more robustly to external stimuli may gain a growth and survival advantage which may possibly promote clonal evolution. Here we report an intra-clonal diversity in response to CD19 engagement in CLL cells identified by using a non-conventional flow cytometric analysis. Monoclonal anti-CD19 antibody rapidly induced cellular aggregation of CLL cells identified as a high side scatters lymphoid population (HSLP). The percentage of the HSLP within the whole CLL population was highly reproducible in individual patients, but was extremely variable between patients (range 0–39.4%, median-19.95%). This inter-individual heterogeneity was attributed to the intrinsic ratio between CD19 responsive and unresponsive CLL subsets and was seen to correlate with time to treatment (TTT), but not with ZAP-70 expression. During a median follow-up period of 45.6 months (n=46), CLL patients with more than 20% of HSLP had a median TTT of 50.7 months from the time of diagnosis while this median has yet not been reached in patients with a lower proportion of HSLP (p=0.019). The rapid induction of cellular aggregation via CD19 was completely abrogated by disruption of the cholesterol-rich plasma membrane rafts by methyl-cyclodextrin, but minimally affected by protein kinase and actin cytoskeleton inhibitors. The relatively ineffective blocking of cellular aggregation by cytoplasmatic signaling inhibitors suggests that this response is mainly attributed to CD19 association with other trans-membrane components, which possibly occurs within the lipid rafts. The CLL sub-population which responded to CD19 clustering expressed higher mRNA levels of c-Myc, and exhibited different morphological features from the non-responsive CLL cells subset. The CD19-responsive subset also correlated with higher CD20 expression and a marked decrease in HSLP was noted in samples taken 24 h after rituximab administration (n=6). As expected, rituximab resulted in a significant decrease in the total CLL counts, with the specific elimination of the HSLP. Comparison of HSLP induction by CD19 in normal B-cells and in leukemic cells from B-type ALL revealed very low levels of HSLP in B-cells from healthy individuals and B-ALL (n=5, range 0.5–2.3%, median-0.9%). Leukemic cells derived from other indolent B-cell lymphoproliferative disorders (non-Hodgkin lymphomas and hairy cell leukemia) had the highest proportion of HSLP of all samples screened (n=8, range 34–60%, median-38.3%). Conclusions:we have identified intra-clonal diversity for CD19-mediated cellular aggregation in CLL which correlates with time to treatment initiation. This phenomenon was completely lipid raft dependent and restricted to a distinct CLL cell subset. The heterogeneous CD19 response may reflect basic differences in the juxta-membrane signaling patterns within the CLL clone. Disclosures:No relevant conflicts of interest to declare.