Presence Of Lipid Rafts Research Articles

Amyloid Precursor Protein Changes Arrangement in a Membrane and Its Structure Depending on the Cholesterol Content.

One of the hallmarks of Alzheimer's disease (AD) is the accumulation of amyloid beta (Aβ) peptides in the brain. The processing of amyloid precursor protein (APP) into Aβ is dependent on the location of APP in the membrane, membrane lipid composition and, possibly, presence of lipid rafts. In this study, we used atomic force microscopy (AFM) to investigate the interaction between transmembrane fragment APP672-726 (corresponding to Aβ1-55) and its amyloidogenic mutant L723P with membranes combining liquid-ordered and liquid-disordered lipid phases. Our results demonstrated that most of the APP672-726 is located either in the liquid-disordered phase or at the boundary between ordered and disordered phases, and hardly ever in rafts. We did not notice any major changes in the domain structure induced by APP672-726. In membranes without cholesterol APP672-726, and especially its amyloidogenic mutant L723P formed annular structures and clusters rising above the membrane. Presence of cholesterol led to the appearance of concave membrane regions up to 2 nm in depth that were deeper for wild type APP672-726. Thus, membrane cholesterol regulates changes in membrane structure and permeability induced by APP that might be connected with further formation of membrane pores.

Formation of lipid raft nanodomains in homogeneous ternary lipid mixture of POPC/DPSM/cholesterol: Theoretical insights

Lipid rafts, in biological membranes, are cholesterol-rich nanodomains that regulate many protein activities and cellular processes. Understanding the formation of the lipid-raft nanodomains helps us elucidate many complex interactions in the cell. In this study, the formation of lipid-raft nanodomains in a ternary palmitoyl-oleoyl-phosphatidylcholine/stearoyl-sphingomyelin/cholesterol (POPC/DPSM/Chol) lipid mixture, the most realistic surrogate model for biological membranes, has been successfully observed for the first time in-silico using microsecond timescale molecular dynamics simulations. The model reveals the formation of cholesterol-induced nanodomains with raft-like characteristics and their underlying mechanism: the cholesterol molecules segregate themselves into cholesterol nanodomains and then enrich the cholesterol-rich domain with sphingomyelin molecules to form a lipid raft thanks to the weak bonding of cholesterol with sphingomyelin. Besides, it is found that the increase in cholesterol concentration enhances the biophysical properties (e.g., bilayer thickness, area per lipid headgroup, and order parameter) of the lipid raft nanodomains. Such findings suggest that the POPC/DPSM/Chol bilayer is a suitable model to fundamentally extend the nanodomain evolution to investigate their lifetime and kinetics as well as to study protein-lipid interaction, protein-protein interaction, and selection of therapeutic molecules in the presence of lipid rafts.

Presence of Androgen Receptor Variant in Neuronal Lipid Rafts.

Fast, nongenomic androgen actions have been described in various cell types, including neurons. However, the receptor mediating this cell membrane–initiated rapid signaling remains unknown. This study found a putative androgen receptor splice variant in a dopaminergic N27 cell line and in several brain regions (substantia nigra pars compacta, entorhinal cortex, and hippocampus) from gonadally intact and gonadectomized (young and middle-aged) male rats. This putative splice variant protein has a molecular weight of 45 kDa and lacks an N-terminal domain, indicating it is homologous to the human AR45 splice variant. Interestingly, AR45 was highly expressed in all brain regions examined. In dopaminergic neurons, AR45 is localized to plasma membrane lipid rafts, a microdomain involved in cellular signaling. Further, AR45 protein interacts with membrane-associated G proteins Gαq and Gαo. Neither age nor hormone levels altered AR45 expression in dopaminergic neurons. These results provide the first evidence of AR45 protein expression in the brain, specifically plasma membrane lipid rafts. AR45 presence in lipid rafts indicates that it may function as a membrane androgen receptor to mediate fast, nongenomic androgen actions.

Lipid-Mediated Clusters of Guest Molecules in Model Membranes and Their Dissolving in the Presence of Lipid Rafts

The clustering of molecules is an important feature of plasma membrane organization. It is challenging to develop methods for quantifying membrane heterogeneities because of their transient nature and small size. Here, we obtained evidence that transient membrane heterogeneities can be frozen at cryogenic temperatures which allows the application of solid-state experimental techniques sensitive to the nanoscale distance range. We employed the pulsed version of electron paramagnetic resonance (EPR) spectroscopy, the electron spin echo (ESE) technique, for spin-labeled molecules in multilamellar lipid bilayers. ESE decays were refined for pure contribution of spin-spin magnetic dipole-dipolar interaction between the labels; these interactions manifest themselves at a nanometer distance range. The bilayers were prepared from different types of saturated and unsaturated lipids and cholesterol (Chol); in all cases, a small amount of guest spin-labeled substances 5-doxyl-stearic-acid (5-DSA) or 3β-doxyl-5α-cholestane (DChl) was added. The local concentration found of 5-DSA and DChl molecules was remarkably higher than the mean concentration in the bilayer, evidencing the formation of lipid-mediated clusters of these molecules. To our knowledge, formation of nanoscale clusters of guest amphiphilic molecules in biological membranes is a new phenomenon suggested only recently. Two-dimensional 5-DSA molecular clusters were found, whereas flat DChl molecules were found to be clustered into stacked one-dimensional structures. These clusters disappear when the Chol content is varied between the boundaries known for lipid raft formation at room temperatures. The room temperature EPR evidenced entrapping of DChl molecules in the rafts.

LAT2, a Lipid Raft Protein That Participates in AKT Phosphorylation in Mantle Cell Lymphoma, Is a Target for Perifosine Chemotherapy

Introduction: Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and sphingolipids and provide an environment for signal transduction proteins which control cell survival and cell death. Altered raft assembly has been implicated in cancer progression. Alkylphospholipids (APL) inhibit the AKT pathway and induce apoptosis of malignant cells but not of their normal counterparts when used at the same concentration. LAT2 (Linker for activation of T-cells family member 2) is a lipid raft component which is disrupted by APLs in leukemic cells. In the present study we evaluated the effect of perifosine, an APL with anticancer activity in humans, on the LAT2 and AKT pathways using a Mantle Cell Lymphoma (MCL) cell line (Granta-519 cells) as a model. We selected MCL cells to study because AKT is constitutively phosphorylated in this disease.Methods: We determined whether perifosine induced apoptosis of unmodified Granta-519 cells using flow cytometry, we measured caspase-3 activity with a synthetic peptide and detected cleaved caspases by western blotting. Then, we demonstrated the participation of LAT2 in AKT signaling. We used a stable knockdown of LAT2 (lentiviral sh-RNA). After transduction, Granta-519 cells were selected using 0.5 µg/mL puromycin for 5 days. Stable LAT2 knockdown reduced LAT2 levels by 90% (Figure 1A) and showed impaired AKT phosphorylation in the absence of ligands (Figure 1B).Results: Perifosine treatment induced apoptosis of Granta-519 cells and the effective dose 50% (ED-50) was 20 μM. Apoptosis was triggered after 6 hours of incubation and activation of the extrinsic and intrinsic pathways of apoptosis was demonstrated. Perifosine decreased LAT2 abundance after 3 hours of treatment, also decreasing its presence in lipid rafts, and led to a decrease in AKT phosphorylation and downstream components of AKT signaling. Moreover, perifosine impaired AKT phosphorylation in the presence of stimuli such as CD40L (330 ng/mL) in Granta-519 cells after a few minutes of incubation, resulting in an effect similar to that of the PI3K inhibitor Wortmannin (1 µM). In vivo studies using Granta-519 subcutaneous xenografts in NSG mice revealed that stable knockdown of LAT2 cells showed reduced tumor growth. sh-RNA CT cells (0.75x106) were injected into the right flank of five mice and the same number of sh-RNA LAT2 cells were injected into the left flank of the same animals. After 3 weeks, the tumor weight (mean + SD) was 0.52 ± 0.37 g for sh-RNA LAT2 tumors compared to 1.41 ± 0.19 g for sh-RNA CT tumors (p=0.0065) (Figure 1C and 1D). The LAT2 content of sh-RNA LAT2 cells and AKT phosphorylation were also reduced (Figure 1E and 1F).Conclusions: These results demonstrate that LAT2 is an important protein for supporting constitutive AKT phosphorylation/activation and downstream signaling in MCL and suggest that inhibition of LAT2 by APLs may have therapeutic applications.Acknowledgments: This research was supported by FAPESP, FINEP, and CNPq. C.H.T. received fellowships from FAPESP Proc. No. 13/07675-3. G.A.F. received a fellowship from CNPq Proc. 150126/2014-0. G.A.F. and C.H.T. contributed equally to this work [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Quantitation and localization of protein molecules in living cells

The structural and functional heterogeneity of cells is exemplified by their polarity, the presence of lipid rafts and caveolae in the plasma membrane, the uneven distribution of receptors on the cell membrane, compartmentalization of messenger molecules and tethering effects of scaffolding molecules, the existence of distinct microcompartments and microdomains within the cell, and of molecules that bind or associate with the cytoskeletal proteins, tubulin and actin. These features imply that the major determinants of cell function, the protein molecules, are assembled in microdomains and interact with each other in distinct locations and at precise concentrations. The issues of protein localization and quantitation in cells are thus of fundamental importance in cell biology. Information on the localization and quantity profiles of proteins in a cell at a given time, together with data from single-molecule studies, will lead to a better understanding of cell functioning. It is proposed that a new term, precision cell biology, should be coined for this kind of cell biology. However, direct demonstration of the spatial relationships between interacting proteins and accurate determination of the quantities of interacting protein molecules in live cells are not easy, and convenient and efficient methods are lacking. Although recent improvements in technologies, such as super-resolution fluorescence microscopy and high-pressure cryoelectron microscopy have greatly facilitated progress in this direction, further studies are needed to satisfy the requirements.

Role of Key Aromatic Residues in the Ligand-binding Domain of α7 Nicotinic Receptors in the Agonist Action of β-Amyloid

Soluble β-amyloid (Aβ) resides in certain regions of the brain at or near picomolar concentration, rising in level during the prodromic stage of Alzheimer disease. Recently, we identified the homomeric α7 nicotinic acetylcholine receptor (α7-nAChR) as one possible functional target for picomolar Aβ. This study was aimed at addressing which residues in α7-nAChRs potentially interact with Aβ to regulate the presynaptic function of this receptor. Site-directed mutagenesis was carried out to study the key aromatic residues in the mouse α7-nAChR agonist-binding pocket. Mutations of tyrosine188 resulted in a decrease in activation of presynaptic α7-nAChRs by ACh and Aβ but with no change in response to nicotine, indicating the critical role of Tyr-188 in presynaptic regulation by Aβ. Coimmunoprecipitation additionally revealed direct binding of Aβ to α7-nAChRs and to the Tyr-188 mutant receptor. In contrast, mutations of Tyr-195 in α7-nAChR led to decreased activation by nicotine without apparent effects on ACh- or Aβ-induced responses. Agonist-induced responses of Tyr-93 mutant α7-nAChRs indicated possible interactions of nicotine and Aβ with its hydroxyl group, but there was no change in presynaptic responses after mutation of Trp-149. All of the mutants were shown to be expressed on the plasma membrane using cell surface labeling. Together, these results directly demonstrate an essential role for the aromatic residue Tyr-188 as a key component in the agonist binding domain for the activation of α7-nAChRs by Aβ.

Lipid Rafts and Cytoskeletal Proteins in Placental Microvilli Membranes from Preeclamptic and IUGR Pregnancies

Intrauterine growth restriction (IUGR) and preeclampsia (PE) are leading causes of perinatal and maternal morbidity and mortality. Previously we reported the expression of lipid rafts in classical microvillous membrane (MVM) and light microvillous membrane (LMVM), two subdomains in apical membrane from the human placental syncytiotrophoblast (hSTB), which constitute the epithelium responsible for maternal-fetal transport. Here the aim was to study the raft and cytoskeletal proteins from PE and IUGR. Microdomains from MVM and LMVM were tested with raft markers (placental alkaline phosphatase, lipid ganglioside, and annexin 2) and a nonraft marker (hTf-R). No changes were detected with those markers in whole purified apical membranes in normal, PE, and IUGR pregnancies; however, their patterns of distribution in lipid rafts were different in PE and IUGR. Cholesterol depletion modified their segregation, confirming their presence in lipid rafts, although unlike normal placenta, in these pathologies there is only one type of microdomain. Additionally, the cytoskeleton proteins actin, ezrin, and cytokeratin-7 showed clear differences between normal and pathological membranes. Cytokeratin-7 expression decreased to 50% in PE, and the distribution between LMVM and MVM (~43 and 57%, respectively) changed in both PE and IUGR, in contrast with the asymmetrical enrichment obtained in normal LMVM (~62%). In conclusion, lipid rafts from IUGR and PE have different features compared to rafts from normal placentae, and this is associated with alterations in the expression and distribution of cytoskeletal proteins.

Impact of KCNE subunits on KCNQ1 (Kv7.1) channel membrane surface targeting

The KCNQ1 (Kv7.1) channel plays an important role in cardiovascular physiology. Cardiomyocytes co-express KCNQ1 with KCNE1-5 proteins. KCNQ1 may co-associate with multiple KCNE regulatory subunits to generate different biophysically and pharmacologically distinct channels. Increasing evidence indicates that the location and targeting of channels are important determinants of their function. In this context, the presence of K(+) channels in sphingolipid-cholesterol-enriched membrane microdomains (lipid rafts) is under investigation. Lipid rafts are important for cardiovascular functioning. We aimed to determine whether KCNE subunits modify the localization and targeting of KCNQ1 channels in lipid rafts microdomains. HEK-293 cells were transiently transfected with KCNQ1 and KCNE1-5, and their traffic and presence in lipid rafts were analyzed. Only KCNQ1 and KCNE3, when expressed alone, co-localized in raft fractions. In addition, while KCNE2 and KCNE5 notably stained the cell surface, KCNQ1 and the rest of the KCNEs showed strong intracellular retention. KCNQ1 targets multiple membrane surface microdomains upon association with KCNE peptides. Thus, while KCNQ1/KCNE1 and KCNQ1/KCNE2 channels target lipid rafts, KCNQ1 associated with KCNE3-5 did not. Channel membrane dynamics, analyzed by fluorescence recovery after photobleaching (FRAP) experiments, further supported these results. In conclusion, the trafficking and targeting pattern of KCNQ1 can be influenced by its association with KCNEs. Since KCNQ1 is crucial for cardiovascular physiology, the temporal and spatial regulations that different KCNE subunits may confer to the channels could have a dramatic impact on membrane electrical activity and putative endocrine regulation.

IL-2 Induces Conformational Changes in Its Preassembled Receptor Core, Which Then Migrates in Lipid Raft and Binds to the Cytoskeleton Meshwork

While interleukin (IL)-2 clearly initiates the sequential assembly of its soluble receptor fragments (sIL-2R) in vitro (with sIL-2Rα first, sIL-2Rβ second, and sγc last), the assembly mechanism of full-length subunits (IL-2R) at the surface of living lymphocytes remains to be elucidated. Here we demonstrate by fluorescence cross-correlated spectroscopy that native IL-2Rβ and γc assemble spontaneously at the surface of living human leukemia T cells (Kit-225 cell line) in the absence of IL-2 and with 1:1 stoichiometry. The dissociation constant of the membrane-embedded IL-2Rβ/γc complex is measured in situ. Förster fluorescence resonance energy transfer analyzed by confocal microscopy of transfected COS-7 cells between combination pairs of various-length receptor chain constructions, using green fluorescent protein derivatives as cytoplasmic carboxy-terminal extensions, showed that IL-2Rβ:ECFP and γc:EYFP bind each other through their extracellular domains, and that IL-2 binding brings their transmembrane domains 30 Å closer together. These observations demonstrate that IL-2Rβ/γc heterodimers are preformed and that their cytoplasmic domains, carrying Janus kinase (Jak) 1 and Jak3, are pulled and tethered together on cytokine binding, triggering signaling transduction.IL-2 binding stabilizes IL-2/IL-2R complexes in membrane nanodomains that promote Jak1/Jak3 phosphorylation. The complexes then interact with the cytoskeleton, which slows receptor diffusion (as measured by fluorescence cross-correlated spectroscopy) and promotes STAT (signal transducer and activator of transcription) 5 phosphorylation. Separation of IL-2-activated receptors from Triton-lysed cells in detergent-resistant membrane nanodomains by ultracentrifugation on a sucrose gradient confirmed their presence in lipid rafts. The release of the IL-2-activated receptor from cytochalasin-treated cells and the IL-2-induced recruitment of actin and tubulin, analyzed by immunoprecipitation, confirmed that the activated receptor interacts with the cytoskeleton. Although IL-2Rα (the third chain that gives the IL-2Rβ/γc receptor core its high affinity for IL-2) is highly expressed at the cell surface and mainly clustered in membrane microdomains at the surface of Kit-225 cells, the few free IL-2Rα present bind last to the IL-2/IL-2Rβ/γc complex and lock IL-2 to its binding site for prolonged action, promoting signal amplification.

Immunoliposomes That Target Endothelium In Vitro Are Dependent on Lipid Raft Formation

Lipid rafts are plasma membrane microdomains rich in cholesterol, sphingolipids, and cell surface receptors. Recent studies demonstrated the upregulation and localization of two receptors, intercellular cell adhesion molecule-1 (ICAM, CD54) and endothelial leukocyte adhesion molecule-1 (E-selectin, CD64E), within lipid raft microdomains of inflamed or injured endothelial cells (ECs). We hypothesized that the localization of ICAM and E-selectin within lipid rafts may be essential for drug delivery vehicles labeled with antibodies against ICAM (aICAM) and E-selectin (aE-selectin). To eliminate localization of cell surface receptors, ECs were treated with a cholesterol depleting drug, methyl-β-cyclodextrin. We also tested if antibody mobility and the ratio of aICAM to aE-selectin on immunoliposomes influenced binding to lipid-raft-depleted cells. Liposomes were prepared from either 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC, C(18:1), T(m) = -20 °C) or 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC, C(16:0), T(m) = 42 °C) which are in the liquid crystalline and gel phase at 37 °C, respectively. Mobility and the aICAM:aE-selectin ratio influenced cellular binding only when lipid rafts form. In the absence of lipid rafts, cellular binding of both DOPC and DPPC immunoliposomes was reduced to the nonspecific binding level. These results, which were obtained under static conditions, suggest that the presence of lipid rafts in ECs is critical for targeted drug delivery.

Single-Molecule Investigation of the Influence Played by Lipid Rafts on Ion Transport and Dynamic Features of the Pore-Forming Alamethicin Oligomer

In this experimental work we employed single-molecule electrical recordings on alamethicin oligomers inserted in lipid bilayers made of brain sphingomyelin (bSM), palmitoyloleoylphosphatidylcholine (POPC) and cholesterol (chol) to unravel novel aspects regarding lipid raft interactions with pore-forming peptides. We probed the effect of lipid rafts on electrical properties of inserted alamethicin oligomers, and our data convincingly prove that the single-channel electrical conductance of various subconductance states of the alamethicin oligomer (1) increases in the presence of raft-containing ternary lipid mixtures (POPC-chol-bSM) compared to cases when bilayers were made of POPC-chol and POPC and (2) decreases in the presence of raft-containing ternary lipid mixtures compared to nonraft ternary mixtures which favor the fluid and liquid ordered phases alone. Our data demonstrate that the presence of lipid rafts leads to a slower association kinetics of alamethicin oligomers, seemingly reflecting a slower lateral diffusion process of such peptide aggregates compared to the case of nonraft, binary lipid mixtures. Furthermore, we show that the electrical capacitance of ternary lipid mixtures (POPC-chol-bSM) decreases in the presence of raft domains by comparison to nonraft binary phases (POPC-chol) or POPC alone, and this could constitute an additional mechanism via which macroscopic electrical manifestations of eukaryotic cells are modulated by the coexistence of gel and fluid domains of the plasma membrane.

Layers of organization of cAMP microdomains in a simple cell

Based on a variety of single-cell measurements, the notion that cAMP microdomains exist in cells is being increasingly embraced. The cellular and molecular underpinnings of this organization are also steadily being revealed. A dependence of Ca(2+)-sensitive ACs (adenylate cyclases) in HEK-293 cells (human embryonic kidney cells) on capacitative Ca(2+) entry is enforced by their presence in lipid rafts and protein-protein interactions. In these cells, many of the participants in the cAMP cascade, including AC, phosphodiesterase 4, cAMP-dependent protein kinase [PKA (protein kinase A)] and protein phosphatase 2A, are now seen to be involved in higher order assemblies. Moreover, the presence of Na(+)/H(+) exchanger 1 in these domains creates a microclimate, protected against global swings in cellular pH. The Ca(2+)-stimulatable AC8, which is targeted to these regions, can sequester calmodulin for its own regulatory purposes. These devices are a sampling of the multiple layers of organization that are in place -- even in a simple cell -- to ensure faithful and economical communication of the cAMP message.

Erlin-1 and erlin-2 are novel members of the prohibitin family of proteins that define lipid-raft-like domains of the ER

Our laboratory was interested in characterizing the molecular composition of non-caveolar lipid rafts. Thus, we generated monoclonal antibodies to lipid raft proteins of human myelomonocytic cells. Two of these proteins, KE04p and C8orf2, were found to be highly enriched in the detergent-insoluble, buoyant fraction of sucrose gradients in a cholesterol-dependent manner. They contain an evolutionarily conserved domain placing them in the prohibitin family of proteins. In contrast to other family members, these two proteins localized to the ER. Furthermore, the extreme N-termini of KE04p and C8orf2 were found to be sufficient for heterologous targeting of GFP to the ER in the absence of classical ER retrieval motifs. We also demonstrate that all prohibitin family members rely on sequences in their extreme N-termini for their distinctive subcellular distributions including the mitochondria, plasma membrane and Golgi vesicles. Owing to their subcellular localization and their presence in lipid rafts, we have named KE04p and C8orf2, ER lipid raft protein (erlin)-1 and erlin-2, respectively. Interestingly, the ER contains relatively low levels of cholesterol and sphingolipids compared with other organelles. Thus, our data support the existence of lipid-raft-like domains within the membranes of the ER.

Involvement of Lipid Raft Domain Signaling in Ex Vivo Maintenance of Hematopoietic Progenitor Cells.

Ex vivo manipulation of hematopoietic stem cell populations has been well documented to result in loss of pluripotentiality, proliferative deficits, and a decreased ability to repopulate post-transplantation. These defects result from a multitude of factors, and here we examine the contribution of lipid raft-localized signaling complexes and pro-survival growth factors, such as hepatocyte growth factor (HGF), in mediation of these defects. Specifically, we have examined the effects of diffused oxygen and hepatocyte growth factor (HGF) on ex vivo culture conditions for preservation of hematopoietic stem cell potentiality. We hypothesize that the high level of diffused oxygen in traditional cell culture systems represents a cellular stress leading to differentiation and loss of potential. We further hypothesize that this effect can be mitigated by the pro-survival factor HGF. Human umbilical cord blood derived mononuclear cells were first isolated by a ficoll density gradient followed by immunomagnetic depletion of mature cell lineages. These lineage depleted cells (Lin-) were cultured on fibronectin-coated plates in serum free media and kept in a 37° humidified atmosphere. We supplemented the cultures with stem cell factor (SCF), Flt-3, thrombopoietin (TPO), and HGF, and then manipulated the available oxygen for the cultured cells between 18% (ambient) and 2–3% (hypoxic). The cells were harvested at various time points (24h, 48h, 72h, and 96h) and assayed for lineage cell markers in conjunction with surrogate markers of lipid raft formation by flow cytometry. Preservation of phenotypic cell surface markers such as CD34, CD133, and CD31 were assayed, as well as the presentation of c-met, the receptor for HGF. Results were validated by immunohistochemistry and confocal microscopy for colocalization of receptor-raft complexes. We found that total lipid raft expression was upregulated in hypoxic conditions versus ambient, with a significant increase in a population of cells that highly expressed lipid raft markers. In addition, we found that cells cultured in the presence of HGF in ambient conditions showed a decrease in the presence of lipid rafts with a trend toward lower expression in those cells cultured with HGF. This population of cells demonstrated a specific preservation of CD34+ expressing cells, and decreased incidence of apoptosis in all compartment surveyed. Functional assays of colony-forming ability, SCID-repopulating activity, and competitive repopulation were also assayed, to correlate the upregulation of lipid raft domain presentation with relevant stem cell ability. Current focus of this investigation centers on the identification of the specific receptor signaling complexes associated with these raft domains.

Prothymosin alpha-receptor associates with lipid rafts in PHA-stimulated lymphocytes

Lipid rafts are specialized plasma membrane microdomains in which glycosphingolipids and cholesterol are major structural components. Their relative insolubility to nonionic detergents is the most widely used method to purify these structures. Several signalling proteins are associated with these microdomains in T lymphocytes, including receptors for growth factors and cytokines. ProTalpha is a highly conserved and widely distributed protein whose physiological functions remain elusive. In previous works we identified, by means of affinity cross-linking, affinity chromatography and fluorescence microscopy, a set of binding proteins for ProTalpha in human lymphoblasts. Now, this work goes deeply in that ProTalpha receptor description revealing, by different experimental approaches, its presence in lipid rafts. Moreover, our results fit a model in which a tyrosine phosphorylation signalling cascade confined to rafts is initiated upon ProTalpha receptor recognition, which represents an important and promising finding in the research for elucidating the molecular mechanisms underlying the immunomodulatory functions of ProTalpha.

Gramicidin D and Dithiothreitol Effects on Erythrocyte Exovesiculation

The use of either diphenylhexatriene, trimethylamino-diphenylhexatriene, or heptadecyl-hydroxycoumarin (C17-HC) allows, simultaneously and with the same molecule, the induction of erythrocyte exovesiculation and labeling of the released vesicles with the fluorescent probe. This method was used to evaluate gramicidin D (a channel-forming peptide) and dithiothreitol (a reducing agent) effects on the human erythrocytes vesiculation process. The release of cholesterol and phospholipids in exovesicles at longer incubation times was only detectable in the presence of gramicidin or dithiothreitol. When C17-HC was used to induce the vesiculation, the presence of gramicidin or dithiothreitol lead to a drastic decrease on the [phospholipids]/[cholesterol] ratio. However, in the samples with dithiothreitol, this variation did not result in the expectable decrease of membrane fluidity. These effects can be related with the presence of lipid rafts, the transbilayer lipids reorientation induced by gramicidin or dithiothreitol, and the cholesterol-dependent gramicidin channels inactivation.

Proteomic Analysis of CLL-Cell Lipid Rafts Reveals Differential Expression of Raft-Associated ATP Synthase.

In chronic lymphocytic leukaemia (CLL) the intracellular signalling response that follows B-cell receptor (BCR) ligation varies between cases. This capacity to signal is linked to established prognostic groups: clones possessing relatively unmutated VH genes (UM-CLL) generally retain the ability to signal, whilst somatically hypermutated clones (M-CLL) are mostly unresponsive. As it is known that proteins associated with lipid rafts are important for the initiation of BCR signalling, we hypothesised that differences in the protein composition of rafts could account for the heterogeneity of BCR responses between UM- and M-CLL clones. Lipid rafts were isolated by density gradient centrifugation of CLL-cell lysates. The buoyant fraction thus obtained was enriched for Lyn kinase and ganglioside GM1, whilst lacking CD45, features consistent with the presence of lipid rafts. Lipid rafts were isolated from 12 CLL clones possessing variable degrees of VH somatic hypermutation and of BCR responsiveness. Raft fractions were subjected to SDS-PAGE and raft-associated proteins visualised by staining with Coomassie Blue. This staining revealed multiple bands with a similar distribution between cases. However case-to-case heterogeneity in the intensity of some protein bands was observed. These differences are likely to reflect true variation in protein expression since equal amounts of lysate were used in each experiment (100 μg of protein/lane). 21 of the visualised protein bands were excised, subjected to tryptic digestion and analysed using MALDI-TOF mass spectrometry. 14 proteins were positively identified with a high degree of confidence with the majority of these being cytoskeletal and structural proteins. One protein in particular was variably expressed in the rafts of different clones and was identified as the F1 subunit of ATP synthase; a finding confirmed by immunoblotting of raft fractions. This differential expression of raft-associated ATP synthase in lipid rafts was associated with the extent of VH gene mutation but not with BCR responsiveness. In five of six UM-CLL clones (VH<5%), raft-associated ATP synthase was detectable, whilst only two of six M-CLL clones (VH>5%) expressed raft-associated ATP synthase. Flow cytometric assessment revealed a uniformly weak expression of ATP synthase on the cell surface of all clones tested. Therefore in those cases lacking raft-associated ATP synthase the protein was present on the cell surface, but excluded from lipid rafts. ATP synthase was initially described as a mitochondrial protein, but has subsequently been identified both on the surface of hepatocytes where it controls cellular lipoprotein uptake, and the surface of lymphocytes where it functions as a ligand for natural killer cells. The role of cell surface ATP synthase in CLL cells is unclear but the presence of this protein in the rafts of somatically unmutated clones could be associated with the poor prognosis of patients in this subgroup and therefore warrants further investigation.

Lipid raft-enriched stem cell-like keratinocytes in the epidermis, hair follicles and sinus tracts in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a disease, that causes considerable morbidity in patients. A histological hallmark of the disorder is the formation of sinus tracts in the dermis and the subcutis. Biologically, they represent a poorly understood phenomenon involving the infiltrative growth of proliferating non-malignant keratinocytes. Lipid domains in plasma membranes (lipid rafts) play a role in the function of growth factors and are suspected of having a pathogenic role in cell migration and invasive growth. Using HS as a model, the presence of lipid rafts was studied using cholera toxin conjugated with FITC (CTx-FITC) and antibeta1 integrin (CD29)-CyChrome conjugate fluorescence staining of unfixed and acetone-fixed cryostat sections of lesional and paralesional skin samples. The double-labeled skin samples were observed in the confocal laser-scanning fluorescence microscope. Samples were obtained from five patients with HS. The lesional epidermis of HS contained three populations of keratinocytes: CD29(bright)CTx(dim), CD29(dim)CTx(bright) and a third hitherto unseen population containing double-positive CD29(bright)CTx(bright) cells. The CD29(bright)CTx(dim) population resembles the earlier described epidermal stem-like cells, while the CD29(dim)CTx(bright) basal keratinocytes overlap with the transit-amplifying cell pool. The new population of double-positive CD29(bright)CTx(bright) cells was localized on the slopes of the papillas, focally in the suprabasal epidermal layers, in some hair follicles and in the majority of sinus tracts. Such double-positive cells have not previously been encountered by us in normal epidermis and hair follicles. Using HS as a model, it is suggested that the keratinocytes involved in sinus tract formation are CD29(bright)CTx(bright) cells. Owing to the physical proximity of the cells, it is hypothesised that the described CD29(bright)CTx(bright) cells result from an increased expression of CD29 on the CTx(bright) cells. It is likely that the double-positive CD29(bright)CTx(bright) cells emerge due to the influence of local inflammatory cytokines. Sinus tract formation may represent an aberrant epidermal repair response executed by the activated CD29(bright)CTx(bright) keratinocytes capable of non-malignant infiltrative growth in the dermis and subcutis.

Oxidized LAT and Hyporesponsiveness

Correct localization to lipid rafts is essential for LAT (linker for activation of T cells)-mediated signaling through the T cell antigen receptor (TCR). Under conditions of chronic oxidative stress, T cells are hyporesponsive to stimuli. For example, in rheumatoid arthritis (RA), T cell insensitivity is thought to contribute to the disorder. Grinhuis et al . found that LAT localization in T cells is perturbed during chronic oxidative stress and results in decreased signaling outputs. LAT isolated from T cells treated with an inhibitor of glutathione (GSH) synthesis exhibited faster migration than LAT from untreated T cells under nonreducing conditions in gel electrophoresis. This suggested that LAT might form intramolecular disulfide bonds under conditions of chronic oxidative stress. Mutational analysis of Cys residues on LAT revealed that C117S or C26/29S mutants were partially insensitive to chronic oxidative stress (presumably because they could not become oxidized and form disulfide bridges), as shown by their continued presence in lipid rafts and by the ability of cells expressing these mutants to stimulate gene expression from interleukin-2 promoter-driven reporter genes. Oxidative stress also greatly reduced the TCR- and LAT-dependent activation of the transcription factors AP-1, NFAT, and NF-κB. However, expression of oxidation-insensitive LAT mutants in T cells partially reversed this inhibition. The authors suggest that the transmembrane helix of LAT might be sufficiently disrupted by chronic oxidative conditions such that LAT cannot remain localized to the lipid rafts. Other data suggested that disulfide bonds might form between Cys117 and probably Cys29. The data suggest that T cell hyporesponsiveness in chronic oxidative conditions results, in part, from improper localization of wild-type LAT, and thus ineffective signaling. For an excellent review on the function of the LAT protein, see the review by Wange . S. I. Gringhuis, E. A. M. Papendrecht-van der Voort, A. Leow, E. W. N. Levarht, F. C. Breedveld, C. L. Verweij, Effect of redox balance alterations on cellular localization of LAT and downstream T-cell receptor signaling pathways. Mol. Cell. Biol. 22 , 400-411 (2002). [Abstract] [Full Text] R. L. Wange, LAT, the linker for activation of T cells: A bridge between T cell-specific and general signaling pathways. Science's STKE (2000), http://www.stke.org/cgi/content/full/OC_sigtrans;2000/63/re1. [Abstract] [Full Text]