Lipid rafts in T cell signalling and disease

PubMed Central

Jury, Elizabeth C.; Flores-Borja, Fabian; Kabouridis, Panagiotis S.

2007-01-01

Lipid rafts is a blanket term used to describe distinct areas in the plasma membrane rich in certain lipids and proteins and which are thought to perform diverse functions. A large number of studies report on lipid rafts having a key role in receptor signalling and activation of lymphocytes. In T cells, lipid raft involvement was demonstrated in the early steps during T cell receptor (TCR) stimulation. Interestingly, recent evidence has shown that signalling in these domains differs in T cells isolated from patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Here, we discuss these findings and explore the potential of lipid rafts as targets for the development of a new class of agents to downmodulate immune responses and for the treatment of autoimmune diseases. PMID:17890113

Cytoplasmic remodeling of erythrocyte raft lipids during infection by the human malaria parasite Plasmodium falciparum

PubMed Central

Murphy, Sean C.; Fernandez-Pol, Sebastian; Chung, Paul H.; Prasanna Murthy, S. N.; Milne, Stephen B.; Salomao, Marcela; Brown, H. Alex; Lomasney, Jon W.; Mohandas, Narla

2007-01-01

Studies of detergent-resistant membrane (DRM) rafts in mature erythrocytes have facilitated identification of proteins that regulate formation of endovacuolar structures such as the parasitophorous vacuolar membrane (PVM) induced by the malaria parasite Plasmodium falciparum. However, analyses of raft lipids have remained elusive because detergents interfere with lipid detection. Here, we use primaquine to perturb the erythrocyte membrane and induce detergent-free buoyant vesicles, which are enriched in cholesterol and major raft proteins flotillin and stomatin and contain low levels of cytoskeleton, all characteristics of raft microdomains. Lipid mass spectrometry revealed that phosphatidylethanolamine and phosphatidylglycerol are depleted in endovesicles while phosphoinositides are highly enriched, suggesting raft-based endovesiculation can be achieved by simple (non–receptor-mediated) mechanical perturbation of the erythrocyte plasma membrane and results in sorting of inner leaflet phospholipids. Live-cell imaging of lipid-specific protein probes showed that phosphatidylinositol (4,5) bisphosphate (PIP2) is highly concentrated in primaquine-induced vesicles, confirming that it is an erythrocyte raft lipid. However, the malarial PVM lacks PIP2, although another raft lipid, phosphatidylserine, is readily detected. Thus, different remodeling/sorting of cytoplasmic raft phospholipids may occur in distinct endovacuoles. Importantly, erythrocyte raft lipids recruited to the invasion junction by mechanical stimulation may be remodeled by the malaria parasite to establish blood-stage infection. PMID:17526861

Analysis of Cd44-Containing Lipid Rafts

PubMed Central

Oliferenko, Snezhana; Paiha, Karin; Harder, Thomas; Gerke, Volker; Schwärzler, Christoph; Schwarz, Heinz; Beug, Hartmut; Günthert, Ursula; Huber, Lukas A.

1999-01-01

CD44, the major cell surface receptor for hyaluronic acid (HA), was shown to localize to detergent-resistant cholesterol-rich microdomains, called lipid rafts, in fibroblasts and blood cells. Here, we have investigated the molecular environment of CD44 within the plane of the basolateral membrane of polarized mammary epithelial cells. We show that CD44 partitions into lipid rafts that contain annexin II at their cytoplasmic face. Both CD44 and annexin II were released from these lipid rafts by sequestration of plasma membrane cholesterol. Partition of annexin II and CD44 to the same type of lipid rafts was demonstrated by cross-linking experiments in living cells. First, when CD44 was clustered at the cell surface by anti-CD44 antibodies, annexin II was recruited into the cytoplasmic leaflet of CD44 clusters. Second, the formation of intracellular, submembranous annexin II–p11 aggregates caused by expression of a trans-dominant mutant of annexin II resulted in coclustering of CD44. Moreover, a frequent redirection of actin bundles to these clusters was observed. These basolateral CD44/annexin II–lipid raft complexes were stabilized by addition of GTPγS or phalloidin in a semipermeabilized and cholesterol-depleted cell system. The low lateral mobility of CD44 in the plasma membrane, as assessed with fluorescent recovery after photobleaching (FRAP), was dependent on the presence of plasma membrane cholesterol and an intact actin cytoskeleton. Disruption of the actin cytoskeleton dramatically increased the fraction of CD44 which could be recovered from the light detergent-insoluble membrane fraction. Taken together, our data indicate that in mammary epithelial cells the vast majority of CD44 interacts with annexin II in lipid rafts in a cholesterol-dependent manner. These CD44-containing lipid microdomains interact with the underlying actin cytoskeleton. PMID:10459018

Isolation and analysis of membrane lipids and lipid rafts in common carp (Cyprinus carpio L.).

PubMed

Brogden, Graham; Propsting, Marcus; Adamek, Mikolaj; Naim, Hassan Y; Steinhagen, Dieter

2014-03-01

Cell membranes act as an interface between the interior of the cell and the exterior environment and facilitate a range of essential functions including cell signalling, cell structure, nutrient uptake and protection. It is composed of a lipid bilayer with integrated proteins, and the inner leaflet of the lipid bilayer comprises of liquid ordered (Lo) and liquid disordered (Ld) domains. Lo microdomains, also named as lipid rafts are enriched in cholesterol, sphingomyelin and certain types of proteins, which facilitate cell signalling and nutrient uptake. Lipid rafts have been extensively researched in mammals and the presence of functional lipid rafts was recently demonstrated in goldfish, but there is currently very little knowledge about their composition and function in fish. Therefore a protocol was established for the analysis of lipid rafts and membranous lipids in common carp (Cyprinus carpio L.) tissues. Twelve lipids were identified and analysed in the Ld domain of the membrane with the most predominant lipids found in all tissues being; triglycerides, cholesterol, phosphoethanolamine and phosphatidylcholine. Four lipids were identified in lipid rafts in all tissues analysed, triglycerides (33-62%) always found in the highest concentration followed by cholesterol (24-32%), phosphatidylcholine and sphingomyelin. Isolation of lipid rafts was confirmed by identifying the presence of the lipid raft associated protein flotillin, present at higher concentrations in the detergent resistant fraction. The data provided here build a lipid library of important carp tissues as a baseline for further studies into virus entry, protein trafficking or environmental stress analysis. Copyright © 2013 Elsevier Inc. All rights reserved.

Lipid rafts are disrupted in mildly inflamed intestinal microenvironments without overt disruption of the epithelial barrier.

PubMed

Bowie, Rachel V; Donatello, Simona; Lyes, Clíona; Owens, Mark B; Babina, Irina S; Hudson, Lance; Walsh, Shaun V; O'Donoghue, Diarmuid P; Amu, Sylvie; Barry, Sean P; Fallon, Padraic G; Hopkins, Ann M

2012-04-15

Intestinal epithelial barrier disruption is a feature of inflammatory bowel disease (IBD), but whether barrier disruption precedes or merely accompanies inflammation remains controversial. Tight junction (TJ) adhesion complexes control epithelial barrier integrity. Since some TJ proteins reside in cholesterol-enriched regions of the cell membrane termed lipid rafts, we sought to elucidate the relationship between rafts and intestinal epithelial barrier function. Lipid rafts were isolated from Caco-2 intestinal epithelial cells primed with the proinflammatory cytokine interferon-γ (IFN-γ) or treated with methyl-β-cyclodextrin as a positive control for raft disruption. Rafts were also isolated from the ilea of mice in which colitis had been induced in conjunction with in vivo intestinal permeability measurements, and lastly from intestinal biopsies of ulcerative colitis (UC) patients with predominantly mild or quiescent disease. Raft distribution was analyzed by measuring activity of the raft-associated enzyme alkaline phosphatase and by performing Western blot analysis for flotillin-1. Epithelial barrier integrity was estimated by measuring transepithelial resistance in cytokine-treated cells or in vivo permeability to fluorescent dextran in colitic mice. Raft and nonraft fractions were analyzed by Western blotting for the TJ proteins occludin and zonula occludens-1 (ZO-1). Our results revealed that lipid rafts were disrupted in IFN-γ-treated cells, in the ilea of mice with subclinical colitis, and in UC patients with quiescent inflammation. This was not associated with a clear pattern of occludin or ZO-1 relocalization from raft to nonraft fractions. Significantly, a time-course study in colitic mice revealed that disruption of lipid rafts preceded the onset of increased intestinal permeability. Our data suggest for the first time that lipid raft disruption occurs early in the inflammatory cascade in murine and human colitis and, we speculate, may contribute to

Evidence that the respiratory syncytial virus polymerase complex associates with lipid rafts in virus-infected cells: a proteomic analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

McDonald, Terence P.; Pitt, Andrew R.; Brown, Gaie

2004-12-05

The interaction between the respiratory syncytial virus (RSV) polymerase complex and lipid rafts was examined in HEp2 cells. Lipid-raft membranes were prepared from virus-infected cells and their protein content was analysed by Western blotting and mass spectrometry. This analysis revealed the presence of the N, P, L, M2-1 and M proteins. However, these proteins appeared to differ from one another in their association with these structures, with the M2-1 protein showing a greater partitioning into raft membranes compared to that of the N, P or M proteins. Determination of the polymerase activity profile of the gradient fractions revealed that 95%more » of the detectable viral enzyme activity was associated with lipid-raft membranes. Furthermore, analysis of virus-infected cells by confocal microscopy suggested an association between these proteins and the raft-lipid, GM1. Together, these results provide evidence that the RSV polymerase complex is able to associate with lipid rafts in virus-infected cells.« less

Analysis of lipid raft molecules in the living brain slices.

PubMed

Kotani, Norihiro; Nakano, Takanari; Ida, Yui; Ito, Rina; Hashizume, Miki; Yamaguchi, Arisa; Seo, Makoto; Araki, Tomoyuki; Hojo, Yasushi; Honke, Koichi; Murakoshi, Takayuki

2017-08-24

Neuronal plasma membrane has been thought to retain a lot of lipid raft components which play important roles in the neural function. Although the biochemical analyses of lipid raft using brain tissues have been extensively carried out in the past 20 years, many of their experimental conditions do not coincide with those of standard neuroscience researches such as neurophysiology and neuropharmacology. Hence, the physiological methods for lipid raft analysis that can be compatible with general neuroscience have been required. Herein, we developed a system to physiologically analyze ganglioside GM1-enriched lipid rafts in brain tissues using the "Enzyme-Mediated Activation of Radical Sources (EMARS)" method that we reported (Kotani N. et al. Proc. Natl. Acad. Sci. U S A 105, 7405-7409 (2008)). The EMARS method was applied to acute brain slices prepared from mouse brains in aCSF solution using the EMARS probe, HRP-conjugated cholera toxin subunit B, which recognizes ganglioside GM1. The membrane molecules present in the GM1-enriched lipid rafts were then labeled with fluorescein under the physiological condition. The fluorescein-tagged lipid raft molecules called "EMARS products" distributed differentially among various parts of the brain. On the other hand, appreciable differences were not detected among segments along the longitudinal axis of the hippocampus. We further developed a device to label the lipid raft molecules in acute hippocampal slices under two different physiological conditions to detect dynamics of the lipid raft molecules during neural excitation. Using this device, several cell membrane molecules including Thy1, known as a lipid raft resident molecule in neurons, were confirmed by the EMARS method in living hippocampal slices. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effect of glycyrrhetinic acid on lipid raft model at the air/water interface.

PubMed

Sakamoto, Seiichi; Uto, Takuhiro; Shoyama, Yukihiro

2015-02-01

To investigate an interfacial behavior of the aglycon of glycyrrhizin (GC), glycyrrhetinic acid (GA), with a lipid raft model consisting of equimolar ternary mixtures of N-palmitoyl sphingomyelin (PSM), dioleoylphosphatidylcholine (DOPC), and cholesterol (CHOL), Langmuir monolayer techniques were systematically conducted. Surface pressure (π)-molecular area (A) and surface potential (ΔV)-A isotherms showed that the adsorbed GA at the air/water interface was desorbed into the bulk upon compression of the lipid monolayer. In situ morphological analysis by Brewster angle microscopy and fluorescence microscopy revealed that the raft domains became smaller as the concentrations of GA in the subphase (CGA) increased, suggesting that GA promotes the formation of fluid networks related to various cellular processes via lipid rafts. In addition, ex situ morphological analysis by atomic force microscopy revealed that GA interacts with lipid raft by lying down at the surface. Interestingly, the distinctive striped regions were formed at CGA=5.0 μM. This phenomenon was observed to be induced by the interaction of CHOL with adsorbed GA and is involved in the membrane-disrupting activity of saponin and its aglycon. A quantitative comparison of GA with GC (Sakamoto et al., 2013) revealed that GA interacts more strongly with the raft model than GC in the monolayer state. Various biological activities of GA are known to be stronger than those of GC. This fact allows us to hypothesize that differences in the interactions of GA/GC with the model monolayer correlate to their degree of exertion for numerous activities. Copyright © 2014 Elsevier B.V. All rights reserved.

In Situ Visualization of Lipid Raft Domains by Fluorescent Glycol Chitosan Derivatives.

PubMed

Jiang, Yao-Wen; Guo, Hao-Yue; Chen, Zhan; Yu, Zhi-Wu; Wang, Zhifei; Wu, Fu-Gen

2016-07-05

Lipid rafts are highly ordered small microdomains mainly composed of glycosphingolipids, cholesterol, and protein receptors. Optically distinguishing lipid raft domains in cell membranes would greatly facilitate the investigations on the structure and dynamics of raft-related cellular behaviors, such as signal transduction, membrane transport (endocytosis), adhesion, and motility. However, current strategies about the visualization of lipid raft domains usually suffer from the low biocompatibility of the probes, invasive detection, or ex situ observation. At the same time, naturally derived biomacromolecules have been extensively used in biomedical field and their interaction with cells remains a long-standing topic since it is closely related to various fundamental studies and potential applications. Herein, noninvasive visualization of lipid raft domains in model lipid bilayers (supported lipid bilayers and giant unilamellar vesicles) and live cells was successfully realized in situ using fluorescent biomacromolecules: the fluorescein isothiocyanate (FITC)-labeled glycol chitosan molecules. We found that the lipid raft domains in model or real membranes could be specifically stained by the FITC-labeled glycol chitosan molecules, which could be attributed to the electrostatic attractive interaction and/or hydrophobic interaction between the probes and the lipid raft domains. Since the FITC-labeled glycol chitosan molecules do not need to completely insert into the lipid bilayer and will not disturb the organization of lipids, they can more accurately visualize the raft domains as compared with other fluorescent dyes that need to be premixed with the various lipid molecules prior to the fabrication of model membranes. Furthermore, the FITC-labeled glycol chitosan molecules were found to be able to resist cellular internalization and could successfully visualize rafts in live cells. The present work provides a new way to achieve the imaging of lipid rafts and also

Implications of lipid raft disintegration: enhanced anti-inflammatory macrophage phenotype.

PubMed

Cuschieri, Joseph

2004-08-01

Lipid rafts are membrane microdomains characterized by an enriched cholesterol environment and appear to serve as a platform for signaling. Their role within the macrophage during endotoxin exposure is unknown. THP-1 cells were subjected to lipopolysaccharide stimulation with or without methyl-beta-cyclodextrin (MbetaCD) pretreatment, a cholesterol depleting agent. Cell surface expression of toll-like receptor-4 (TLR4) and platelet-activating factor receptor (PAFr) was determined by flow cytometry. Membrane receptor components and activation of the mitogen-activated protein kinases (MAPK) was determined from lipid raft and cellular protein by immunoblot. Inflammatory mediator production was determined from harvested supernatants by enzyme-linked immunosorbent assay. Surface expression of TLR4 and PAFr was not affected by MbetaCD. Lipopolysaccharide stimulation led to TLR4 mobilization to lipid rafts, MAPK activation, and inflammatory mediator production. Pretreatment with MbetaCD did not affect TLR4 mobilization to lipid rafts, but did result in lost lipid raft expression of the PAFr coupled G-protein, Galpha1. MbetaCD treatment led to selective attenuation of MAPK activation through ERK 1/2. This dysregulated signaling was associated with attenuated production of tumor necrosis factor-alpha, but increased production of interleukin-10. Lipid raft disintegration results in lost expression of Galpha1, dysregulated MAPK signaling, and selective anti-inflammatory mediator production. Therefore, modulation of lipid raft cholesterol content may represent a potential mechanism for regulation of macrophage phenotypic differentiation. Copyright 2004 Elsevier Inc.

Lipid rafts are essential for peroxisome biogenesis in HepG2 cells.

PubMed

Woudenberg, Jannes; Rembacz, Krzysztof P; Hoekstra, Mark; Pellicoro, Antonella; van den Heuvel, Fiona A J; Heegsma, Janette; van Ijzendoorn, Sven C D; Holzinger, Andreas; Imanaka, Tsuneo; Moshage, Han; Faber, Klaas Nico

2010-08-01

Peroxisomes are particularly abundant in the liver and are involved in bile salt synthesis and fatty acid metabolism. Peroxisomal membrane proteins (PMPs) are required for peroxisome biogenesis [e.g., the interacting peroxisomal biogenesis factors Pex13p and Pex14p] and its metabolic function [e.g., the adenosine triphosphate-binding cassette transporters adrenoleukodystrophy protein (ALDP) and PMP70]. Impaired function of PMPs is the underlying cause of Zellweger syndrome and X-linked adrenoleukodystrophy. Here we studied for the first time the putative association of PMPs with cholesterol-enriched lipid rafts and their function in peroxisome biogenesis. Lipid rafts were isolated from Triton X-100-lysed or Lubrol WX-lysed HepG2 cells and analyzed for the presence of various PMPs by western blotting. Lovastatin and methyl-beta-cyclodextrin were used to deplete cholesterol and disrupt lipid rafts in HepG2 cells, and this was followed by immunofluorescence microscopy to determine the subcellular location of catalase and PMPs. Cycloheximide was used to inhibit protein synthesis. Green fluorescent protein-tagged fragments of PMP70 and ALDP were analyzed for their lipid raft association. PMP70 and Pex14p were associated with Triton X-100-resistant rafts, ALDP was associated with Lubrol WX-resistant rafts, and Pex13p was not lipid raft-associated in HepG2 cells. The minimal peroxisomal targeting signals in ALDP and PMP70 were not sufficient for lipid raft association. Cholesterol depletion led to dissociation of PMPs from lipid rafts and impaired sorting of newly synthesized catalase and ALDP but not Pex14p and PMP70. Repletion of cholesterol to these cells efficiently reestablished the peroxisomal sorting of catalase but not ALDP. Human PMPs are differentially associated with lipid rafts independently of the protein homology and/or their functional interaction. Cholesterol is required for peroxisomal lipid raft assembly and peroxisome biogenesis.

Mesoscale organization of domains in the plasma membrane - beyond the lipid raft.

PubMed

Lu, Stella M; Fairn, Gregory D

2018-04-01

The plasma membrane is compartmentalized into several distinct regions or domains, which show a broad diversity in both size and lifetime. The segregation of lipids and membrane proteins is thought to be driven by the lipid composition itself, lipid-protein interactions and diffusional barriers. With regards to the lipid composition, the immiscibility of certain classes of lipids underlies the "lipid raft" concept of plasmalemmal compartmentalization. Historically, lipid rafts have been described as cholesterol and (glyco)sphingolipid-rich regions of the plasma membrane that exist as a liquid-ordered phase that are resistant to extraction with non-ionic detergents. Over the years the interest in lipid rafts grew as did the challenges with studying these nanodomains. The term lipid raft has fallen out of favor with many scientists and instead the terms "membrane raft" or "membrane nanodomain" are preferred as they connote the heterogeneity and dynamic nature of the lipid-protein assemblies. In this article, we will discuss the classical lipid raft hypothesis and its limitations. This review will also discuss alternative models of lipid-protein interactions, annular lipid shells, and larger membrane clusters. We will also discuss the mesoscale organization of plasmalemmal domains including visible structures such as clathrin-coated pits and caveolae.

Involvement of glycosphingolipid-enriched lipid rafts in inflammatory responses.

PubMed

Iwabuchi, Kazuhisa

2015-01-01

Glycosphingolipids (GSLs) are membrane components consisting of hydrophobic ceramide and hydrophilic sugar moieties. GSLs cluster with cholesterol in cell membranes to form GSL-enriched lipid rafts. Biochemical analyses have demonstrated that GSL-enriched lipid rafts contain several kinds of transducer molecules, including Src family kinases. Among the GSLs, lactosylceramide (LacCer, CDw17) can bind to various microorganisms, is highly expressed on the plasma membranes of human phagocytes, and forms lipid rafts containing the Src family tyrosine kinase Lyn. LacCer-enriched lipid rafts mediate immunological and inflammatory reactions, including superoxide generation, chemotaxis, and non-opsonic phagocytosis. Therefore, LacCer-enriched membrane microdomains are thought to function as pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs) expressed on microorganisms. LacCer also serves as a signal transduction molecule for functions mediated by CD11b/CD18-integrin (αM/β2-integrin, CR3, Mac-1), as well as being associated with several key cellular processes. LacCer recruits PCKα/ε and phospholipase A2 to stimulate PECAM-1 expression in human monocytes and their adhesion to endothelial cells, as well as regulating β1-integrin clustering and endocytosis on cell surfaces. This review describes the organizational and inflammation-related functions of LacCer-enriched lipid rafts.

Compartmentalized cAMP Signaling Associated With Lipid Raft and Non-raft Membrane Domains in Adult Ventricular Myocytes.

PubMed

Agarwal, Shailesh R; Gratwohl, Jackson; Cozad, Mia; Yang, Pei-Chi; Clancy, Colleen E; Harvey, Robert D

2018-01-01

Aim: Confining cAMP production to discrete subcellular locations makes it possible for this ubiquitous second messenger to elicit unique functional responses. Yet, factors that determine how and where the production of this diffusible signaling molecule occurs are incompletely understood. The fluid mosaic model originally proposed that signal transduction occurs through random interactions between proteins diffusing freely throughout the plasma membrane. However, it is now known that the movement of membrane proteins is restricted, suggesting that the plasma membrane is segregated into distinct microdomains where different signaling proteins can be concentrated. In this study, we examined what role lipid raft and non-raft membrane domains play in compartmentation of cAMP signaling in adult ventricular myocytes. Methods and Results: The freely diffusible fluorescence resonance energy transfer-based biosensor Epac2-camps was used to measure global cytosolic cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. We found that β-adrenergic receptors, which are expressed in lipid raft and non-raft domains, produce cAMP responses near the plasma membrane that are distinctly different from those produced by E-type prostaglandin receptors, which are expressed exclusively in non-raft domains. We also found that there are differences in basal cAMP levels associated with lipid raft and non-raft domains, and that this can be explained by differences in basal adenylyl cyclase activity associated with each of these membrane environments. In addition, we found evidence that phosphodiesterases 2, 3, and 4 work together in regulating cAMP activity associated with both lipid raft and non-raft domains, while phosphodiesterase 3 plays a more prominent role in the bulk cytoplasmic compartment. Conclusion: These results suggest that different membrane

Docosahexaenoic acid alters Gsα localization in lipid raft and potentiates adenylate cyclase.

PubMed

Zhu, Zhuoran; Tan, Zhoubin; Li, Yan; Luo, Hongyan; Hu, Xinwu; Tang, Ming; Hescheler, Jürgen; Mu, Yangling; Zhang, Lanqiu

2015-01-01

Supplementation with docosahexaenoic acid (DHA), an ω-3 polyunsaturated fatty acid (PUFA), recently has become popular for the amelioration of depression; however the molecular mechanism of DHA action remains unclear. The aim of this study was to investigate the mechanism underlying the antidepressant effect of DHA by evaluating Gsα localization in lipid raft and the activity of adenylate cyclase in an in vitro glioma cell model. Lipid raft fractions from C6 glioma cells treated chronically with DHA were isolated by sucrose gradient ultracentrifugation. The content of Gsα in lipid raft was analyzed by immunoblotting and colocalization of Gsα with lipid raft was subjected to confocal microscopic analysis. The intracellular cyclic adenosine monophosphate (cAMP) level was determined by cAMP immunoassay kit. DHA decreased the amount of Gsα in lipid raft, whereas whole cell lysate Gsα was not changed. Confocal microscopic analysis demonstrated that colocalization of Gsα with lipid raft was decreased, whereas DHA increased intracellular cAMP accumulation in a dose-dependent manner. Interestingly, we found that DHA increased the lipid raft level, instead of disrupting it. The results of this study suggest that DHA may exert its antidepressant effect by translocating Gsα from lipid raft and potentiating the activity of adenylate cyclase. Importantly, the reduced Gsα in lipid raft by DHA is independent of disruption of lipid raft. Overall, the study provides partial preclinical evidence supporting a safe and effective therapy using DHA for depression. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparative lipidomics and proteomics analysis of platelet lipid rafts using different detergents.

PubMed

Rabani, Vahideh; Davani, Siamak; Gambert-Nicot, Ségolène; Meneveau, Nicolas; Montange, Damien

2016-11-01

Lipid rafts play a pivotal role in physiological functions of platelets. Their isolation using nonionic mild detergents is considered as the gold standard method, but there is no consensual detergent for lipid raft studies. We aimed to investigate which detergent is the most suitable for lipid raft isolation from platelet membrane, based on lipidomics and proteomics analysis. Platelets were obtained from healthy donors. Twelve sucrose fractions were extracted by three different detergents, namely Brij 35, Lubrol WX, and Triton X100, at 0.05% and 1%. After lipidomics analysis and determination of fractions enriched in cholesterol (Ch) and sphingomyelin (SM), proteomics analysis was performed. Lipid rafts were mainly observed in 1-4 fractions, and non-rafts were distributed on 5-12 fractions. Considering the concentration of Ch and SM, Lubrol WX 1% and Triton X100 1% were more suitable detergents as they were able to isolate lipid raft fractions that were more enriched than non-raft fractions. By proteomics analysis, overall, 822 proteins were identified in platelet membrane. Lipid raft fractions isolated with Lubrol WX 0.05% and Triton X100 1% contained mainly plasma membrane proteins. However, only Lubrol WX 0.05 and 1% and Triton X100 1% were able to extract non-denaturing proteins with more than 10 transmembrane domains. Our results suggest that Triton X100 1% is the most suitable detergent for global lipid and protein studies on platelet plasma membrane. However, the detergent should be adapted if investigation of an association between specific proteins and lipid rafts is planned.

Severe Alterations in Lipid Composition of Frontal Cortex Lipid Rafts from Parkinson’s Disease and Incidental Parkinson’s Disease

PubMed Central

Fabelo, Noemí; Martín, Virginia; Santpere, Gabriel; Marín, Raquel; Torrent, Laia; Ferrer, Isidre; Díaz, Mario

2011-01-01

Lipid rafts are cholesterol- and sphingomyelin-enriched microdomains that provide a highly saturated and viscous physicochemical microenvironment to promote protein–lipid and protein–protein interactions. We purified lipid rafts from human frontal cortex from normal, early motor stages of Parkinson’s disease (PD) and incidental Parkinson’s disease (iPD) subjects and analyzed their lipid composition. We observed that lipid rafts from PD and iPD cortices exhibit dramatic reductions in their contents of n-3 and n-6 long-chain polyunsaturated fatty acids, especially docosahexaenoic acid (22:6-n3) and arachidonic acid (20:4n-6). Also, saturated fatty acids (16:0 and 18:0) were significantly higher than in control brains. Paralleling these findings, unsaturation and peroxidability indices were considerably reduced in PD and iPD lipid rafts. Lipid classes were also affected in PD and iPD lipid rafts. Thus, phosphatidylserine and phosphatidylinositol were increased in PD and iPD, whereas cerebrosides and sulfatides and plasmalogen levels were considerably diminished. Our data pinpoint a dramatic increase in lipid raft order due to the aberrant biochemical structure in PD and iPD and indicate that these abnormalities of lipid rafts in the frontal cortex occur at early stages of PD pathology. The findings correlate with abnormal lipid raft signaling and cognitive decline observed during the development of these neurodegenerative disorders. PMID:21717034

Proteomic Analysis of Lipid Raft-Like Detergent-Resistant Membranes of Lens Fiber Cells.

PubMed

Wang, Zhen; Schey, Kevin L

2015-12-01

Plasma membranes of lens fiber cells have high levels of long-chain saturated fatty acids, cholesterol, and sphingolipids-key components of lipid rafts. Thus, lipid rafts are expected to constitute a significant portion of fiber cell membranes and play important roles in lens biology. The purpose of this study was to characterize the lens lipid raft proteome. Quantitative proteomics, both label-free and iTRAQ methods, were used to characterize lens fiber cell lipid raft proteins. Detergent-resistant, lipid raft membrane (DRM) fractions were isolated by sucrose gradient centrifugation. To confirm protein localization to lipid rafts, protein sensitivity to cholesterol removal by methyl-β-cyclodextrin was quantified by iTRAQ analysis. A total of 506 proteins were identified in raft-like detergent-resistant membranes. Proteins identified support important functions of raft domains in fiber cells, including trafficking, signal transduction, and cytoskeletal organization. In cholesterol-sensitivity studies, 200 proteins were quantified and 71 proteins were strongly affected by cholesterol removal. Lipid raft markers flotillin-1 and flotillin-2 and a significant fraction of AQP0, MP20, and AQP5 were found in the DRM fraction and were highly sensitive to cholesterol removal. Connexins 46 and 50 were more abundant in nonraft fractions, but a small fraction of each was found in the DRM fraction and was strongly affected by cholesterol removal. Quantification of modified AQP0 confirmed that fatty acylation targeted this protein to membrane raft domains. These data represent the first comprehensive profile of the lipid raft proteome of lens fiber cells and provide information on membrane protein organization in these cells.

Cholesterol Regulates μ-Opioid Receptor-Induced β-Arrestin 2 Translocation to Membrane Lipid RaftsS⃞

PubMed Central

Qiu, Yu; Wang, Yan; Chen, Hong-Zhuan; Loh, Horace H.

2011-01-01

μ-Opioid receptor (OPRM1) is mainly localized in lipid raft microdomains but internalizes through clathrin-dependent pathways. Our previous studies demonstrated that disruption of lipid rafts by cholesterol-depletion reagent blocked the agonist-induced internalization of OPRM1 and G protein-dependent signaling. The present study demonstrated that reduction of cholesterol level decreased and culturing cells in excess cholesterol increased the agonist-induced internalization and desensitization of OPRM1, respectively. Further analyses indicated that modulation of cellular cholesterol level did not affect agonist-induced receptor phosphorylation but did affect membrane translocation of β-arrestins. The translocation of β-arrestins was blocked by cholesterol reduction, and the effect could be reversed by incubating with cholesterol. OptiPrep gradient separation of lipid rafts revealed that excess cholesterol retained more receptors in lipid raft domains and facilitated the recruitment of β-arrestins to these microdomains upon agonist activation. Moreover, excess cholesterol could evoke receptor internalization and protein kinase C-independent extracellular signal-regulated kinases activation upon morphine treatment. Therefore, these results suggest that cholesterol not only can influence OPRM1 localization in lipid rafts but also can effectively enhance the recruitment of β-arrestins and thereby affect the agonist-induced trafficking and agonist-dependent signaling of OPRM1. PMID:21518774

Proteomic Analysis of Lipid Raft-Like Detergent-Resistant Membranes of Lens Fiber Cells

PubMed Central

Wang, Zhen; Schey, Kevin L.

2015-01-01

Purpose Plasma membranes of lens fiber cells have high levels of long-chain saturated fatty acids, cholesterol, and sphingolipids—key components of lipid rafts. Thus, lipid rafts are expected to constitute a significant portion of fiber cell membranes and play important roles in lens biology. The purpose of this study was to characterize the lens lipid raft proteome. Methods Quantitative proteomics, both label-free and iTRAQ methods, were used to characterize lens fiber cell lipid raft proteins. Detergent-resistant, lipid raft membrane (DRM) fractions were isolated by sucrose gradient centrifugation. To confirm protein localization to lipid rafts, protein sensitivity to cholesterol removal by methyl-β-cyclodextrin was quantified by iTRAQ analysis. Results A total of 506 proteins were identified in raft-like detergent-resistant membranes. Proteins identified support important functions of raft domains in fiber cells, including trafficking, signal transduction, and cytoskeletal organization. In cholesterol-sensitivity studies, 200 proteins were quantified and 71 proteins were strongly affected by cholesterol removal. Lipid raft markers flotillin-1 and flotillin-2 and a significant fraction of AQP0, MP20, and AQP5 were found in the DRM fraction and were highly sensitive to cholesterol removal. Connexins 46 and 50 were more abundant in nonraft fractions, but a small fraction of each was found in the DRM fraction and was strongly affected by cholesterol removal. Quantification of modified AQP0 confirmed that fatty acylation targeted this protein to membrane raft domains. Conclusions These data represent the first comprehensive profile of the lipid raft proteome of lens fiber cells and provide information on membrane protein organization in these cells. PMID:26747763

Antidepressants Accumulate in Lipid Rafts Independent of Monoamine Transporters to Modulate Redistribution of the G Protein, Gαs.

PubMed

Erb, Samuel J; Schappi, Jeffrey M; Rasenick, Mark M

2016-09-16

Depression is a significant public health problem for which currently available medications, if effective, require weeks to months of treatment before patients respond. Previous studies have shown that the G protein responsible for increasing cAMP (Gαs) is increasingly localized to lipid rafts in depressed subjects and that chronic antidepressant treatment translocates Gαs from lipid rafts. Translocation of Gαs, which shows delayed onset after chronic antidepressant treatment of rats or of C6 glioma cells, tracks with the delayed onset of therapeutic action of antidepressants. Because antidepressants appear to specifically modify Gαs localized to lipid rafts, we sought to determine whether structurally diverse antidepressants accumulate in lipid rafts. Sustained treatment of C6 glioma cells, which lack 5-hydroxytryptamine transporters, showed marked concentration of several antidepressants in raft fractions, as revealed by increased absorbance and by mass fingerprint. Closely related molecules without antidepressant activity did not concentrate in raft fractions. Thus, at least two classes of antidepressants accumulate in lipid rafts and effect translocation of Gαs to the non-raft membrane fraction, where it activates the cAMP-signaling cascade. Analysis of the structural determinants of raft localization may both help to explain the hysteresis of antidepressant action and lead to design and development of novel substrates for depression therapeutics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

O-glycans direct selectin ligands to lipid rafts on leukocytes

PubMed Central

Shao, Bojing; Yago, Tadayuki; Setiadi, Hendra; Wang, Ying; Mehta-D’souza, Padmaja; Fu, Jianxin; Crocker, Paul R.; Rodgers, William; Xia, Lijun; McEver, Rodger P.

2015-01-01

Palmitoylated cysteines typically target transmembrane proteins to domains enriched in cholesterol and sphingolipids (lipid rafts). P-selectin glycoprotein ligand-1 (PSGL-1), CD43, and CD44 are O-glycosylated proteins on leukocytes that associate with lipid rafts. During inflammation, they transduce signals by engaging selectins as leukocytes roll in venules, and they move to the raft-enriched uropods of polarized cells upon chemokine stimulation. It is not known how these glycoproteins associate with lipid rafts or whether this association is required for signaling or for translocation to uropods. Here, we found that loss of core 1-derived O-glycans in murine C1galt1−/− neutrophils blocked raft targeting of PSGL-1, CD43, and CD44, but not of other glycosylated proteins, as measured by resistance to solubilization in nonionic detergent and by copatching with a raft-resident sphingolipid on intact cells. Neuraminidase removal of sialic acids from wild-type neutrophils also blocked raft targeting. C1galt1−/− neutrophils or neuraminidase-treated neutrophils failed to activate tyrosine kinases when plated on immobilized anti–PSGL-1 or anti-CD44 F(ab′)2. Furthermore, C1galt1−/− neutrophils incubated with anti–PSGL-1 F(ab′)2 did not generate microparticles. In marked contrast, PSGL-1, CD43, and CD44 moved normally to the uropods of chemokine-stimulated C1galt1−/− neutrophils. These data define a role for core 1-derived O-glycans and terminal sialic acids in targeting glycoprotein ligands for selectins to lipid rafts of leukocytes. Preassociation of these glycoproteins with rafts is required for signaling but not for movement to uropods. PMID:26124096

O-glycans direct selectin ligands to lipid rafts on leukocytes.

PubMed

Shao, Bojing; Yago, Tadayuki; Setiadi, Hendra; Wang, Ying; Mehta-D'souza, Padmaja; Fu, Jianxin; Crocker, Paul R; Rodgers, William; Xia, Lijun; McEver, Rodger P

2015-07-14

Palmitoylated cysteines typically target transmembrane proteins to domains enriched in cholesterol and sphingolipids (lipid rafts). P-selectin glycoprotein ligand-1 (PSGL-1), CD43, and CD44 are O-glycosylated proteins on leukocytes that associate with lipid rafts. During inflammation, they transduce signals by engaging selectins as leukocytes roll in venules, and they move to the raft-enriched uropods of polarized cells upon chemokine stimulation. It is not known how these glycoproteins associate with lipid rafts or whether this association is required for signaling or for translocation to uropods. Here, we found that loss of core 1-derived O-glycans in murine C1galt1(-/-) neutrophils blocked raft targeting of PSGL-1, CD43, and CD44, but not of other glycosylated proteins, as measured by resistance to solubilization in nonionic detergent and by copatching with a raft-resident sphingolipid on intact cells. Neuraminidase removal of sialic acids from wild-type neutrophils also blocked raft targeting. C1galt1(-/-) neutrophils or neuraminidase-treated neutrophils failed to activate tyrosine kinases when plated on immobilized anti-PSGL-1 or anti-CD44 F(ab')2. Furthermore, C1galt1(-/-) neutrophils incubated with anti-PSGL-1 F(ab')2 did not generate microparticles. In marked contrast, PSGL-1, CD43, and CD44 moved normally to the uropods of chemokine-stimulated C1galt1(-/-) neutrophils. These data define a role for core 1-derived O-glycans and terminal sialic acids in targeting glycoprotein ligands for selectins to lipid rafts of leukocytes. Preassociation of these glycoproteins with rafts is required for signaling but not for movement to uropods.

Isolation and characterization of lipid rafts in Emiliania huxleyi: a role for membrane microdomains in host-virus interactions.

PubMed

Rose, Suzanne L; Fulton, James M; Brown, Christopher M; Natale, Frank; Van Mooy, Benjamin A S; Bidle, Kay D

2014-04-01

Coccolithoviruses employ a suite of glycosphingolipids (GSLs) to successfully infect the globally important coccolithophore Emiliania huxleyi. Lipid rafts, chemically distinct membrane lipid microdomains that are enriched in GSLs and are involved in sensing extracellular stimuli and activating signalling cascades through protein-protein interactions, likely play a fundamental role in host-virus interactions. Using combined lipidomics, proteomics and bioinformatics, we isolated and characterized the lipid and protein content of lipid rafts from control E. huxleyi cells and those infected with EhV86, the type strain for Coccolithovirus. Lipid raft-enriched fractions were isolated and purified as buoyant, detergent-resistant membranes (DRMs) in OptiPrep density gradients. Transmission electron microscopy of vesicle morphology, polymerase chain reaction amplification of the EhV major capsid protein gene and immunoreactivity to flotillin antisera served as respective physical, molecular and biochemical markers. Subsequent lipid characterization of DRMs via high performance liquid chromatography-triple quadrapole mass spectrometry revealed four distinct GSL classes. Parallel proteomic analysis confirmed flotillin as a major lipid raft protein, along with a variety of proteins affiliated with host defence, programmed cell death and innate immunity pathways. The detection of an EhV86-encoded C-type lectin-containing protein confirmed that infection occurs at the interface between lipid rafts and cellular stress/death pathways via specific GSLs and raft-associated proteins. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Lipid Raft Redox Signaling: Molecular Mechanisms in Health and Disease

PubMed Central

Zhou, Fan; Katirai, Foad

2011-01-01

Abstract Lipid rafts, the sphingolipid and cholesterol-enriched membrane microdomains, are able to form different membrane macrodomains or platforms upon stimulations, including redox signaling platforms, which serve as a critical signaling mechanism to mediate or regulate cellular activities or functions. In particular, this raft platform formation provides an important driving force for the assembling of NADPH oxidase subunits and the recruitment of other related receptors, effectors, and regulatory components, resulting, in turn, in the activation of NADPH oxidase and downstream redox regulation of cell functions. This comprehensive review attempts to summarize all basic and advanced information about the formation, regulation, and functions of lipid raft redox signaling platforms as well as their physiological and pathophysiological relevance. Several molecular mechanisms involving the formation of lipid raft redox signaling platforms and the related therapeutic strategies targeting them are discussed. It is hoped that all information and thoughts included in this review could provide more comprehensive insights into the understanding of lipid raft redox signaling, in particular, of their molecular mechanisms, spatial-temporal regulations, and physiological, pathophysiological relevances to human health and diseases. Antioxid. Redox Signal. 15, 1043–1083. PMID:21294649

Generation of stable lipid raft microdomains in the enterocyte brush border by selective endocytic removal of non-raft membrane.

PubMed

Danielsen, E Michael; Hansen, Gert H

2013-01-01

The small intestinal brush border has an unusually high proportion of glycolipids which promote the formation of lipid raft microdomains, stabilized by various cross-linking lectins. This unique membrane organization acts to provide physical and chemical stability to the membrane that faces multiple deleterious agents present in the gut lumen, such as bile salts, digestive enzymes of the pancreas, and a plethora of pathogens. In the present work, we studied the constitutive endocytosis from the brush border of cultured jejunal explants of the pig, and the results indicate that this process functions to enrich the contents of lipid raft components in the brush border. The lipophilic fluorescent marker FM, taken up into early endosomes in the terminal web region (TWEEs), was absent from detergent resistant membranes (DRMs), implying an association with non-raft membrane. Furthermore, neither major lipid raft-associated brush border enzymes nor glycolipids were detected by immunofluorescence microscopy in subapical punctae resembling TWEEs. Finally, two model raft lipids, BODIPY-lactosylceramide and BODIPY-GM1, were not endocytosed except when cholera toxin subunit B (CTB) was present. In conclusion, we propose that constitutive, selective endocytic removal of non-raft membrane acts as a sorting mechanism to enrich the brush border contents of lipid raft components, such as glycolipids and the major digestive enzymes. This sorting may be energetically driven by changes in membrane curvature when molecules move from a microvillar surface to an endocytic invagination.

Aggregation of lipid rafts activates c-met and c-Src in non-small cell lung cancer cells.

PubMed

Zeng, Juan; Zhang, Heying; Tan, Yonggang; Sun, Cheng; Liang, Yusi; Yu, Jinyang; Zou, Huawei

2018-05-30

Activation of c-Met, a receptor tyrosine kinase, induces radiation therapy resistance in non-small cell lung cancer (NSCLC). The activated residual of c-Met is located in lipid rafts (Duhon et al. Mol Carcinog 49:739-49, 2010). Therefore, we hypothesized that disturbing the integrity of lipid rafts would restrain the activation of the c-Met protein and reverse radiation resistance in NSCLC. In this study, a series of experiments was performed to test this hypothesis. NSCLC A549 and H1993 cells were incubated with methyl-β-cyclodextrin (MβCD), a lipid raft inhibitor, at different concentrations for 1 h before the cells were X-ray irradiated. The following methods were used: clonogenic (colony-forming) survival assays, flow cytometry (for cell cycle and apoptosis analyses), immunofluorescence microscopy (to show the distribution of proteins in lipid rafts), Western blotting, and biochemical lipid raft isolation (purifying lipid rafts to show the distribution of proteins in lipid rafts). Our results showed that X-ray irradiation induced the aggregation of lipid rafts in A549 cells, activated c-Met and c-Src, and induced c-Met and c-Src clustering to lipid rafts. More importantly, MβCD suppressed the proliferation of A549 and H1993 cells, and the combination of MβCD and radiation resulted in additive increases in A549 and H1993 cell apoptosis. Destroying the integrity of lipid rafts inhibited the aggregation of c-Met and c-Src to lipid rafts and reduced the expression of phosphorylated c-Met and phosphorylated c-Src in lipid rafts. X-ray irradiation induced the aggregation of lipid rafts and the clustering of c-Met and c-Src to lipid rafts through both lipid raft-dependent and lipid raft-independent mechanisms. The lipid raft-dependent activation of c-Met and its downstream pathways played an important role in the development of radiation resistance in NSCLC cells mediated by c-Met. Further studies are still required to explore the molecular mechanisms of the

Procyanidins can interact with Caco-2 cell membrane lipid rafts: involvement of cholesterol.

PubMed

Verstraeten, Sandra V; Jaggers, Grayson K; Fraga, Cesar G; Oteiza, Patricia I

2013-11-01

Large procyanidins (more than three subunits) are not absorbed at the gastrointestinal tract but could exert local effects through their interactions with membranes. We previously showed that hexameric procyanidins (Hex), although not entering cells, interact with membranes modulating cell signaling and fate. This paper investigated if Hex, as an example of large procyanidins, can selectively interact with lipid rafts which could in part explain its biological actions. This mechanism was studied in both synthetic membranes (liposomes) and Caco-2 cells. Hex promoted Caco-2 cell membrane rigidification and dehydration, effects that were abolished upon cholesterol depletion with methyl-β-cyclodextrin (MCD). Hex prevented lipid raft structure disruption induced by cholesterol depletion/redistribution by MCD or sodium deoxycholate. Supporting the involvement of cholesterol-Hex bonding in Hex interaction with lipid rafts, the absence of cholesterol markedly decreased the capacity of Hex to prevent deoxycholate- and Triton X-100-mediated disruption of lipid raft-like liposomes. Stressing the functional relevance of this interaction, Hex mitigated lipid raft-associated activation of the extracellular signal-regulated kinases (ERK) 1/2. Results support the capacity of a large procyanidin (Hex) to interact with membrane lipid rafts mainly through Hex-cholesterol bondings. Procyanidin-lipid raft interactions can in part explain the capacity of large procyanidins to modulate cell physiology. © 2013 Elsevier B.V. All rights reserved.

Lipid Raft Size and Lipid Mobility in Non-raft Domains Increase during Aging and Are Exacerbated in APP/PS1 Mice Model of Alzheimer's Disease. Predictions from an Agent-Based Mathematical Model

PubMed Central

Santos, Guido; Díaz, Mario; Torres, Néstor V.

2016-01-01

A connection between lipid rafts and Alzheimer's disease has been studied during the last decades. Mathematical modeling approaches have recently been used to correlate the effects of lipid composition changes in the physicochemical properties of raft-like membranes. Here we propose an agent based model to assess the effect of lipid changes in lipid rafts on the evolution and progression of Alzheimer's disease using lipid profile data obtained in an established model of familial Alzheimer's disease. We have observed that lipid raft size and lipid mobility in non-raft domains are two main factors that increase during age and are accelerated in the transgenic Alzheimer's disease mouse model. The consequences of these changes are discussed in the context of neurotoxic amyloid β production. Our agent based model predicts that increasing sterols (mainly cholesterol) and long-chain polyunsaturated fatty acids (LCPUFA) (mainly DHA, docosahexaenoic acid) proportions in the membrane composition might delay the onset and progression of the disease. PMID:27014089

Lipid raft-associated β-adducin is required for PSGL-1-mediated neutrophil rolling on P-selectin.

PubMed

Xu, Tingshuang; Liu, Wenai; Yang, Chen; Ba, Xueqing; Wang, Xiaoguang; Jiang, Yong; Zeng, Xianlu

2015-02-01

Lipid rafts, a liquid-ordered plasma membrane microdomain, are related to cell-surface receptor function. PSGL-1, a major surface receptor protein for leukocyte, also acts as a signaling receptor in leukocyte rolling. To investigate the role of lipid raft in PSGL-1 signaling in human neutrophils, we quantitatively analyzed lipid raft proteome of human promyelocytic leukemia cell line HL-60 cells and identified a lipid raft-associated protein β-adducin. PSGL-1 ligation induced dissociation of the raft-associated protein β-adducin from lipid rafts and actin, as well as phosphorylation of β-adducin, indicating a transient uncoupling of lipid rafts from the actin cytoskeleton. Knockdown of β-adducin greatly attenuated HL-60 cells rolling on P-selectin. We also showed that Src kinase is crucial for PSGL-1 ligation-induced β-adducin phosphorylation and relocation. Taken together, these results show that β-adducin is a pivotal lipid raft-associated protein in PSGL-1-mediated neutrophil rolling on P-selectin. © Society for Leukocyte Biology.

You Sank My Lipid Rafts!

ERIC Educational Resources Information Center

Campbell, Tessa N.

2009-01-01

The plasma membrane is the membrane that serves as a boundary between the interior of a cell and its extracellular environment. Lipid rafts are microdomains within a cellular membrane that possess decreased fluidity due to the presence of cholesterol, glycolipids, and phospholipids containing longer fatty acids. These domains are involved in many…

Lipid raft integrity affects GABAA receptor, but not NMDA receptor modulation by psychopharmacological compounds.

PubMed

Nothdurfter, Caroline; Tanasic, Sascha; Di Benedetto, Barbara; Uhr, Manfred; Wagner, Eva-Maria; Gilling, Kate E; Parsons, Chris G; Rein, Theo; Holsboer, Florian; Rupprecht, Rainer; Rammes, Gerhard

2013-07-01

Lipid rafts have been shown to play an important role for G-protein mediated signal transduction and the function of ligand-gated ion channels including their modulation by psychopharmacological compounds. In this study, we investigated the functional significance of the membrane distribution of NMDA and GABAA receptor subunits in relation to the accumulation of the tricyclic antidepressant desipramine (DMI) and the benzodiazepine diazepam (Diaz). In the presence of Triton X-100, which allowed proper separation of the lipid raft marker proteins caveolin-1 and flotillin-1 from the transferrin receptor, all receptor subunits were shifted to the non-raft fractions. In contrast, under detergent-free conditions, NMDA and GABAA receptor subunits were detected both in raft and non-raft fractions. Diaz was enriched in non-raft fractions without Triton X-100 in contrast to DMI, which preferentially accumulated in lipid rafts. Impairment of lipid raft integrity by methyl-β-cyclodextrine (MβCD)-induced cholesterol depletion did not change the inhibitory effect of DMI at the NMDA receptor, whereas it enhanced the potentiating effect of Diaz at the GABAA receptor at non-saturating concentrations of GABA. These results support the hypothesis that the interaction of benzodiazepines with the GABAA receptor likely occurs outside of lipid rafts while the antidepressant DMI acts on ionotropic receptors both within and outside these membrane microdomains.

Alkyl ether lipids, ion channels and lipid raft reorganization in cancer therapy.

PubMed

Jaffrès, Paul-Alain; Gajate, Consuelo; Bouchet, Ana Maria; Couthon-Gourvès, Hélène; Chantôme, Aurélie; Potier-Cartereau, Marie; Besson, Pierre; Bougnoux, Philippe; Mollinedo, Faustino; Vandier, Christophe

2016-09-01

Synthetic alkyl lipids, such as the ether lipids edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) and ohmline (1-O-hexadecyl-2-O-methyl-rac-glycero-3-β-lactose), are forming a class of antitumor agents that target cell membranes to induce apoptosis and to decrease cell migration/invasion, leading to the inhibition of tumor and metastasis development. In this review, we present the structure-activity relationship of edelfosine and ohmline, and we point out differences and similarities between these two amphiphilic compounds. We also discuss the mechanisms of action of these synthetic alkyl ether lipids (involving, among other structures and molecules, membrane domains, Fas/CD95 death receptor signaling, and ion channels), and highlight a key role for lipid rafts in the underlying process. The reorganization of lipid raft membrane domains induced by these alkyl lipids affects the function of death receptors and ion channels, thus leading to apoptosis and/or inhibition of cancer cell migration. The possible therapeutic use of these alkyl lipids and the clinical perspectives for these lipids in prevention or/and treatment of tumor development and metastasis are also discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

Transcriptional Regulation of T-Cell Lipid Metabolism: Implications for Plasma Membrane Lipid Rafts and T-Cell Function.

PubMed

Robinson, George A; Waddington, Kirsty E; Pineda-Torra, Ines; Jury, Elizabeth C

2017-01-01

It is well established that cholesterol and glycosphingolipids are enriched in the plasma membrane (PM) and form signaling platforms called lipid rafts, essential for T-cell activation and function. Moreover, changes in PM lipid composition affect the biophysical properties of lipid rafts and have a role in defining functional T-cell phenotypes. Here, we review the role of transcriptional regulators of lipid metabolism including liver X receptors α/β, peroxisome proliferator-activated receptor γ, estrogen receptors α/β (ERα/β), and sterol regulatory element-binding proteins in T-cells. These receptors lie at the interface between lipid metabolism and immune cell function and are endogenously activated by lipids and/or hormones. Importantly, they regulate cellular cholesterol, fatty acid, glycosphingolipid, and phospholipid levels but are also known to modulate a broad spectrum of immune responses. The current evidence supporting a role for lipid metabolism pathways in controlling immune cell activation by influencing PM lipid raft composition in health and disease, and the potential for targeting lipid biosynthesis pathways to control unwanted T-cell activation in autoimmunity is reviewed.

Transcriptional Regulation of T-Cell Lipid Metabolism: Implications for Plasma Membrane Lipid Rafts and T-Cell Function

PubMed Central

Robinson, George A.; Waddington, Kirsty E.; Pineda-Torra, Ines; Jury, Elizabeth C.

2017-01-01

It is well established that cholesterol and glycosphingolipids are enriched in the plasma membrane (PM) and form signaling platforms called lipid rafts, essential for T-cell activation and function. Moreover, changes in PM lipid composition affect the biophysical properties of lipid rafts and have a role in defining functional T-cell phenotypes. Here, we review the role of transcriptional regulators of lipid metabolism including liver X receptors α/β, peroxisome proliferator-activated receptor γ, estrogen receptors α/β (ERα/β), and sterol regulatory element-binding proteins in T-cells. These receptors lie at the interface between lipid metabolism and immune cell function and are endogenously activated by lipids and/or hormones. Importantly, they regulate cellular cholesterol, fatty acid, glycosphingolipid, and phospholipid levels but are also known to modulate a broad spectrum of immune responses. The current evidence supporting a role for lipid metabolism pathways in controlling immune cell activation by influencing PM lipid raft composition in health and disease, and the potential for targeting lipid biosynthesis pathways to control unwanted T-cell activation in autoimmunity is reviewed. PMID:29225604

Quantitative Profiling of Brain Lipid Raft Proteome in a Mouse Model of Fragile X Syndrome

PubMed Central

Kalinowska, Magdalena; Castillo, Catherine; Francesconi, Anna

2015-01-01

Fragile X Syndrome, a leading cause of inherited intellectual disability and autism, arises from transcriptional silencing of the FMR1 gene encoding an RNA-binding protein, Fragile X Mental Retardation Protein (FMRP). FMRP can regulate the expression of approximately 4% of brain transcripts through its role in regulation of mRNA transport, stability and translation, thus providing a molecular rationale for its potential pleiotropic effects on neuronal and brain circuitry function. Several intracellular signaling pathways are dysregulated in the absence of FMRP suggesting that cellular deficits may be broad and could result in homeostatic changes. Lipid rafts are specialized regions of the plasma membrane, enriched in cholesterol and glycosphingolipids, involved in regulation of intracellular signaling. Among transcripts targeted by FMRP, a subset encodes proteins involved in lipid biosynthesis and homeostasis, dysregulation of which could affect the integrity and function of lipid rafts. Using a quantitative mass spectrometry-based approach we analyzed the lipid raft proteome of Fmr1 knockout mice, an animal model of Fragile X syndrome, and identified candidate proteins that are differentially represented in Fmr1 knockout mice lipid rafts. Furthermore, network analysis of these candidate proteins reveals connectivity between them and predicts functional connectivity with genes encoding components of myelin sheath, axonal processes and growth cones. Our findings provide insight to aid identification of molecular and cellular dysfunctions arising from Fmr1 silencing and for uncovering shared pathologies between Fragile X syndrome and other autism spectrum disorders. PMID:25849048

Lipid raft regulates the initial spreading of melanoma A375 cells by modulating β1 integrin clustering.

PubMed

Wang, Ruifei; Bi, Jiajia; Ampah, Khamal Kwesi; Zhang, Chunmei; Li, Ziyi; Jiao, Yang; Wang, Xiaoru; Ba, Xueqing; Zeng, Xianlu

2013-08-01

Cell adhesion and spreading require integrins-mediated cell-extracellular matrix interaction. Integrins function through binding to extracellular matrix and subsequent clustering to initiate focal adhesion formation and actin cytoskeleton rearrangement. Lipid raft, a liquid ordered plasma membrane microdomain, has been reported to play major roles in membrane motility by regulating cell surface receptor function. Here, we identified that lipid raft integrity was required for β1 integrin-mediated initial spreading of melanoma A375 cells on fibronectin. We found that lipid raft disruption with methyl-β-cyclodextrin led to the inability of focal adhesion formation and actin cytoskeleton rearrangement by preventing β1 integrin clustering. Furthermore, we explored the possible mechanism by which lipid raft regulates β1 integrin clustering and demonstrated that intact lipid raft could recruit and modify some adaptor proteins, such as talin, α-actinin, vinculin, paxillin and FAK. Lipid raft could regulate the location of these proteins in lipid raft fractions and facilitate their binding to β1 integrin, which may be crucial for β1 integrin clustering. We also showed that lipid raft disruption impaired A375 cell migration in both transwell and wound healing models. Together, these findings provide a new insight for the relationship between lipid raft and the regulation of integrins. Copyright © 2013 Elsevier Ltd. All rights reserved.

T-lymphocyte signalling in systemic lupus erythematosus: a lipid raft perspective

PubMed Central

Jury, EC; Kabouridis, PS

2008-01-01

In the last few years it has become clear that in cells of the immune system, specialized microdomains present in the plasma membrane, called lipid rafts, have been found to play a central role in regulating signalling by immune receptors. Recent studies have looked at whether lipid rafts may be connected to the abnormalities in signalling seen in T lymphocytes isolated from patients with systemic lupus erythematosus (SLE). These early findings show that in SLE T cells, the expression and protein composition of lipid rafts is different when compared with normal T cells. These results also demonstrate changes in the function and localization of critical signalling molecules such as the LCK tyrosine kinase and the CD45 tyrosine phosphatase. PMID:15303567

Clomipramine counteracts lipid raft disturbance due to short-term muscle disuse.

PubMed

Bryndina, Irina G; Shalagina, Maria N; Sekunov, Alexey V; Zefirov, Andrei L; Petrov, Alexey M

2018-01-18

Disuse-induced skeletal muscle dysfunction is a serious consequence of long-term spaceflight, numerous diseases and conditions for which treatment possibilities are still strictly limited. We have previously shown that acute hindlimb suspension (HS)-mediated disuse disrupts membrane lipid rafts in the unloaded muscle. Here, we investigated whether pretreatment of rats with the inhibitor of acid sphingomyelinase, clomipramine (1.25mg/g/day, intramuscularly, for 5days before HS), is able to hinder the loss in lipid raft integrity in response to 12h of HS. Clomipramine pretreatment significantly counteracted the decrease in labeling of the plasma membranes with lipid raft markers (fluorescent cholera toxin B subunit and bodipy-GM1-ganglioside) specifically in the junctional regions of the suspended soleus muscle. This was associated with: a) enhancing raft disrupting potential of exogenous sphingomyelinase in the junctional membranes; b) prevention of both ceramide accumulation and cholesterol loss; c) prevention of decline in nicotinic acetylcholine receptor labeling in the unloaded muscle. Our data suggest that sphingomyelinase-mediated raft disturbance serves as one of the earlier events in HS effects. Copyright © 2017 Elsevier B.V. All rights reserved.

Lipid raft disarrangement as a result of neuropathological progresses: a novel strategy for early diagnosis?

PubMed

Marin, R; Rojo, J A; Fabelo, N; Fernandez, C E; Diaz, M

2013-08-15

Lipid rafts are the preferential site of numerous membrane signaling proteins which are involved in neuronal functioning and survival. These proteins are organized in multiprotein complexes, or signalosomes, in close contact with lipid classes particularly represented in lipid rafts (i.e. cholesterol, sphingolipids and saturated fatty acids), which may contribute to physiological responses leading to neuroprotection. Increasing evidence indicates that alteration of lipid composition in raft structures as a consequence of neuropathologies, such as Alzheimer's disease (AD) and Parkinson's disease (PD), causes a dramatic increase in lipid raft order. These phenomena may correlate with perturbation of signalosome activities, likely contributing to neurodegenerative progression. Interestingly, significant disruption of stable raft microenvironments has been already observed in the first stages of either AD or PD, suggesting that these alterations may represent early events in the neuropathological development. In this regard, the search for biochemical markers, such as specific metabolic products altered in the brain at the first steps of the disease, presently represents an important challenge for early diagnostic strategies. Alterations of these biomarkers may be reflected in either plasma or cerebrospinal fluid, thus representing a potential strategy to predict an accurate diagnosis. We propose that pathologically-linked lipid raft markers may be interesting candidates to be explored at this level, although it has not been studied so far to what extent alteration of different signalosome components may be reflected in peripheral fluids. In this mini-review, we will discuss on relevant aspects of lipid rafts that contribute to the modulation of neuropathological events related to AD and PD. An interesting hypothesis is that anomalies on raft biomarkers measured at peripheral fluids might mirror the lipid raft pathology observed in early stages of AD and PD. Copyright

Myo1c regulates lipid raft recycling to control cell spreading, migration and Salmonella invasion.

PubMed

Brandstaetter, Hemma; Kendrick-Jones, John; Buss, Folma

2012-04-15

A balance between endocytosis and membrane recycling regulates the composition and dynamics of the plasma membrane. Internalization and recycling of cholesterol- and sphingolipid-enriched lipid rafts is an actin-dependent process that is mediated by a specialized Arf6-dependent recycling pathway. Here, we identify myosin1c (Myo1c) as the first motor protein that drives the formation of recycling tubules emanating from the perinuclear recycling compartment. We demonstrate that the single-headed Myo1c is a lipid-raft-associated motor protein that is specifically involved in recycling of lipid-raft-associated glycosylphosphatidylinositol (GPI)-linked cargo proteins and their delivery to the cell surface. Whereas Myo1c overexpression increases the levels of these raft proteins at the cell surface, in cells depleted of Myo1c function through RNA interference or overexpression of a dominant-negative mutant, these tubular transport carriers of the recycling pathway are lost and GPI-linked raft markers are trapped in the perinuclear recycling compartment. Intriguingly, Myo1c only selectively promotes delivery of lipid raft membranes back to the cell surface and is not required for recycling of cargo, such as the transferrin receptor, which is mediated by parallel pathways. The profound defect in lipid raft trafficking in Myo1c-knockdown cells has a dramatic impact on cell spreading, cell migration and cholesterol-dependent Salmonella invasion; processes that require lipid raft transport to the cell surface to deliver signaling components and the extra membrane essential for cell surface expansion and remodeling. Thus, Myo1c plays a crucial role in the recycling of lipid raft membrane and proteins that regulate plasma membrane plasticity, cell motility and pathogen entry.

Lipid rafts sense and direct electric field-induced migration.

PubMed

Lin, Bo-Jian; Tsao, Shun-Hao; Chen, Alex; Hu, Shu-Kai; Chao, Ling; Chao, Pen-Hsiu Grace

2017-08-08

Endogenous electric fields (EFs) are involved in developmental regulation and wound healing. Although the phenomenon is known for more than a century, it is not clear how cells perceive the external EF. Membrane proteins, responding to electrophoretic and electroosmotic forces, have long been proposed as the sensing molecules. However, specific charge modification of surface proteins did not change cell migration motility nor directionality in EFs. Moreover, symmetric alternating current (AC) EF directs cell migration in a frequency-dependent manner. Due to their charge and ability to coalesce, glycolipids are therefore the likely primary EF sensor driving polarization of membrane proteins and intracellular signaling. We demonstrate that detergent-resistant membrane nanodomains, also known as lipid rafts, are the primary response element in EF sensing. The clustering and activation of caveolin and signaling proteins further stabilize raft structure and feed-forward downstream signaling events, such as rho and PI3K activation. Theoretical modeling supports the experimental results and predicts AC frequency-dependent cell and raft migration. Our results establish a fundamental mechanism for cell electrosensing and provide a role in lipid raft mechanotransduction.

Modulation of ileal bile acid transporter (ASBT) activity by depletion of plasma membrane cholesterol: association with lipid rafts

PubMed Central

Annaba, Fadi; Sarwar, Zaheer; Kumar, Pradeep; Saksena, Seema; Turner, Jerrold R.; Dudeja, Pradeep K.; Gill, Ravinder K.; Alrefai, Waddah A.

2016-01-01

Apical sodium-dependent bile acid transporter (ASBT) represents a highly efficient conservation mechanism of bile acids via mediation of their active transport across the luminal membrane of terminal ileum. To gain insight into the cellular regulation of ASBT, we investigated the association of ASBT with cholesterol and sphingolipid-enriched specialized plasma membrane microdomains known as lipid rafts and examined the role of membrane cholesterol in maintaining ASBT function. Human embryonic kidney (HEK)-293 cells stably transfected with human ASBT, human ileal brush-border membrane vesicles, and human intestinal epithelial Caco-2 cells were utilized for these studies. Floatation experiments on Optiprep density gradients demonstrated the association of ASBT protein with lipid rafts. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-β-cyclodextrin (MβCD) significantly reduced the association of ASBT with lipid rafts, which was paralleled by a decrease in ASBT activity in Caco-2 and HEK-293 cells treated with MβCD. The inhibition in ASBT activity by MβCD was blocked in the cells treated with MβCD-cholesterol complexes. Kinetic analysis revealed that MβCD treatment decreased the Vmax of the transporter, which was not associated with alteration in the plasma membrane expression of ASBT. Our study illustrates that cholesterol content of lipid rafts is essential for the optimal activity of ASBT and support the association of ASBT with lipid rafts. These findings suggest a novel mechanism by which ASBT activity may be rapidly modulated by alterations in cholesterol content of plasma membrane and thus have important implications in processes related to maintenance of bile acid and cholesterol homeostasis. PMID:18063707

Myo1c regulates lipid raft recycling to control cell spreading, migration and Salmonella invasion

PubMed Central

Brandstaetter, Hemma; Kendrick-Jones, John; Buss, Folma

2012-01-01

A balance between endocytosis and membrane recycling regulates the composition and dynamics of the plasma membrane. Internalization and recycling of cholesterol- and sphingolipid-enriched lipid rafts is an actin-dependent process that is mediated by a specialized Arf6-dependent recycling pathway. Here, we identify myosin1c (Myo1c) as the first motor protein that drives the formation of recycling tubules emanating from the perinuclear recycling compartment. We demonstrate that the single-headed Myo1c is a lipid-raft-associated motor protein that is specifically involved in recycling of lipid-raft-associated glycosylphosphatidylinositol (GPI)-linked cargo proteins and their delivery to the cell surface. Whereas Myo1c overexpression increases the levels of these raft proteins at the cell surface, in cells depleted of Myo1c function through RNA interference or overexpression of a dominant-negative mutant, these tubular transport carriers of the recycling pathway are lost and GPI-linked raft markers are trapped in the perinuclear recycling compartment. Intriguingly, Myo1c only selectively promotes delivery of lipid raft membranes back to the cell surface and is not required for recycling of cargo, such as the transferrin receptor, which is mediated by parallel pathways. The profound defect in lipid raft trafficking in Myo1c-knockdown cells has a dramatic impact on cell spreading, cell migration and cholesterol-dependent Salmonella invasion; processes that require lipid raft transport to the cell surface to deliver signaling components and the extra membrane essential for cell surface expansion and remodeling. Thus, Myo1c plays a crucial role in the recycling of lipid raft membrane and proteins that regulate plasma membrane plasticity, cell motility and pathogen entry. PMID:22328521

Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells.

PubMed

Morel, Etienne; Ghezzal, Sara; Lucchi, Géraldine; Truntzer, Caroline; Pais de Barros, Jean-Paul; Simon-Plas, Françoise; Demignot, Sylvie; Mineo, Chieko; Shaul, Philip W; Leturque, Armelle; Rousset, Monique; Carrière, Véronique

2018-02-01

Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics. Copyright © 2017 Elsevier B.V. All rights reserved.

Differential insertion of GPI-anchored GFPs into lipid rafts of live cells.

PubMed

Legler, Daniel F; Doucey, Marie-Agnès; Schneider, Pascal; Chapatte, Laurence; Bender, Florent C; Bron, Claude

2005-01-01

Partitioning of proteins in cholesterol and sphingolipid enriched plasma membrane microdomains, called lipid rafts, is critical for many signal transduction and protein sorting events. Although raft partitioning of many signaling molecules remains to be determined, glycosylphosphatidyl-inositol (GPI)-anchored proteins possess high affinity for lipid rafts and are currently exploited as markers to investigate fundamental mechanisms in protein sorting and signal transduction events. In this study, we demonstrate that two recombinant GPI-anchored green fluorescent proteins (GFP-GPIs) that differ in their GPI signal sequence confer distinct localization in plasma membrane microdomains. GFP fused to the GPI signal of the decay accelerating factor GFP-GPI(DAF) partitioned exclusively in lipid rafts, whereas GFP fused to the GPI signal of TRAIL-R3, GFP-GPI(TRAIL-R3), associated only minimally with microdomains. In addition, we investigated the unique ability of purified GFP-GPIs to insert into membrane microdomains of primary lymphocytes. This cell surface painting allows rapid, stable, and functional association of the GPI-anchored proteins with the target cell plasma membrane. The distinct membrane localization of the two GFP-GPIs was observed irrespective of whether the GPI-anchored molecules were painted or transfected. Furthermore, we show that painted GFP-GPI(DAF) was totally dependent on the GPI anchor and that the membrane insertion was increased by the addition of raft-associated lipids such as cholesterol, sphingomyelin, and dipalmitoyl-phosphatidylethanolamine. Thus, this study provides evidence that different GPI signal sequences lead to distinct membrane microdomain localization and that painted GFP-GPI(DAF) serves as an excellent fluorescent marker for lipid rafts in live cells.

Preparation of membrane rafts.

PubMed

Waugh, Mark G; Hsuan, J Justin

2009-01-01

The concept that biological membranes contain microdomains of specialized lipid and protein composition has attracted great attention in recent years. Initially, the focus in the field was very much on the characterization of cholesterol-and sphingolipid-rich plasma membrane microdomains that were resistant to solubilization in the cold non-ionic detergent Triton X-100. Such detergent-insoluble membrane domains were of low buoyant density and could be readily purified on sucrose equilibrium density gradients. The intrinsic buoyancy of the detergent-insoluble domains gave rise to the term "lipid rafts." Cholesterol- and sphingolipid-rich rafts at the plasma membrane have been implicated in a wide range of cellular processes, including pathogen invasion, receptor signaling, and endocytosis. However, work with other non-ionic detergents such as Lubrol WX and Brij-98 has revealed the existence of various raft subtypes with differing lipid compositions and proposed functions. More recently, there has been some focus on isolating lipid rafts from intracellular organelles, in particular membranes from the Golgi-endosomal pathway, where raft lipids have been proposed to function in processes such as the sorting of vesicular cargo and the processing of amyloid precursor protein. While there remains a large degree of controversy surrounding the purity, the physiological importance, and even the existence of different types of lipid rafts in intact cells, the ability to routinely purify such domains has led to significant progress in understanding the functional architecture of biological membranes. We describe a number of widely used methods to prepare rafts, based on early preparations of caveolae by density gradient ultracentrifugation and immunoaffinity precipitation.

Lipid rafts sense and direct electric field-induced migration

PubMed Central

Lin, Bo-jian; Tsao, Shun-hao; Chen, Alex; Hu, Shu-Kai; Chao, Ling

2017-01-01

Endogenous electric fields (EFs) are involved in developmental regulation and wound healing. Although the phenomenon is known for more than a century, it is not clear how cells perceive the external EF. Membrane proteins, responding to electrophoretic and electroosmotic forces, have long been proposed as the sensing molecules. However, specific charge modification of surface proteins did not change cell migration motility nor directionality in EFs. Moreover, symmetric alternating current (AC) EF directs cell migration in a frequency-dependent manner. Due to their charge and ability to coalesce, glycolipids are therefore the likely primary EF sensor driving polarization of membrane proteins and intracellular signaling. We demonstrate that detergent-resistant membrane nanodomains, also known as lipid rafts, are the primary response element in EF sensing. The clustering and activation of caveolin and signaling proteins further stabilize raft structure and feed-forward downstream signaling events, such as rho and PI3K activation. Theoretical modeling supports the experimental results and predicts AC frequency-dependent cell and raft migration. Our results establish a fundamental mechanism for cell electrosensing and provide a role in lipid raft mechanotransduction. PMID:28739955

Association of gamma-secretase with lipid rafts in post-Golgi and endosome membranes.

PubMed

Vetrivel, Kulandaivelu S; Cheng, Haipeng; Lin, William; Sakurai, Takashi; Li, Tong; Nukina, Nobuyuki; Wong, Philip C; Xu, Huaxi; Thinakaran, Gopal

2004-10-22

Alzheimer's disease-associated beta-amyloid peptides (Abeta) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases. There is growing evidence that cholesterol- and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major beta-secretase in neurons is a palmitoylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the gamma-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1(-/-)/PS2(-/-) and NCT(-/-) fibroblasts, gamma-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires gamma-secretase complex assembly. Biochemical evidence shows that subunits of the gamma-secretase complex and three TGN/endosome-resident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1- or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of gamma-secretase in lipid raft microdomains of post-Golgi and endosomes, organelles previously implicated in amyloidogenic processing of APP.

Distinct lipid rafts in subdomains from human placental apical syncytiotrophoblast membranes.

PubMed

Godoy, Valeria; Riquelme, Gloria

2008-01-01

We report on the characteristics of raft domains in the apical membrane from human placental syncytiotrophoblast (hSTB), an epithelium responsible for maternal-fetal exchange. Previously, we described two isolated fractions of the hSTB apical membrane: a classical microvillous membrane (MVM) and a light microvillous membrane (LMVM). Detergent-resistant microdomains (DRMs) from MVM and LMVM were prepared with Triton X-100 followed by flotation in a sucrose gradient and tested by Western and dot blot with raft markers (placental alkaline phosphatase, lipid ganglioside, annexin 2) and transferrin receptor as a nonraft marker. DRMs from both fractions showed a consistent peak for these markers, except that the DRMs from MVM had no annexin 2 mark. Cholesterol depletion modified the segregation in both groups of DRMs. Our results show two distinguishable lipid raft subsets from MVM and LMVM. Additionally, we found significant differences between MVM and LMVM in cholesterol content and in expression of cytoskeletal proteins. MVM is enriched in ezrin and beta-actin; in contrast, cholesterol and cytokeratin-7 are more abundant in LMVM. These differences may explain the distinct properties of the lipid raft subtypes.

Fluorescent probes for lipid rafts: from model membranes to living cells.

PubMed

Klymchenko, Andrey S; Kreder, Rémy

2014-01-16

Membrane microdomains (rafts) remain one of the controversial issues in biophysics. Fluorescent molecular probes, which make these lipid nanostructures visible through optical techniques, are one of the tools currently used to study lipid rafts. The most common are lipophilic fluorescent probes that partition specifically into liquid ordered or liquid disordered phase. Their partition depends on the lipid composition of a given phase, which complicates their use in cellular membranes. A second class of probes is based on environment-sensitive dyes, which partition into both phases, but stain them by different fluorescence color, intensity, or lifetime. These probes can directly address the properties of each separate phase, but their cellular applications are still limited. The present review focuses on summarizing the current state in the field of developing and applying fluorescent molecular probes to study lipid rafts. We highlight an urgent need to develop new probes, specifically adapted for cell plasma membranes and compatible with modern fluorescence microscopy techniques to push the understanding of membrane microdomains forward. Copyright © 2014 Elsevier Ltd. All rights reserved.

Lipid Raft Association Restricts CD44-Ezrin Interaction and Promotion of Breast Cancer Cell Migration

PubMed Central

Donatello, Simona; Babina, Irina S.; Hazelwood, Lee D.; Hill, Arnold D.K.; Nabi, Ivan R.; Hopkins, Ann M.

2012-01-01

Cancer cell migration is an early event in metastasis, the main cause of breast cancer-related deaths. Cholesterol-enriched membrane domains called lipid rafts influence the function of many molecules, including the raft-associated protein CD44. We describe a novel mechanism whereby rafts regulate interactions between CD44 and its binding partner ezrin in migrating breast cancer cells. Specifically, in nonmigrating cells, CD44 and ezrin localized to different membranous compartments: CD44 predominantly in rafts, and ezrin in nonraft compartments. After the induction of migration (either nonspecific or CD44-driven), CD44 affiliation with lipid rafts was decreased. This was accompanied by increased coprecipitation of CD44 and active (threonine-phosphorylated) ezrin-radixin-moesin (ERM) proteins in nonraft compartments and increased colocalization of CD44 with the nonraft protein, transferrin receptor. Pharmacological raft disruption using methyl-β-cyclodextrin also increased CD44-ezrin coprecipitation and colocalization, further suggesting that CD44 interacts with ezrin outside rafts during migration. Conversely, promoting CD44 retention inside lipid rafts by pharmacological inhibition of depalmitoylation virtually abolished CD44-ezrin interactions. However, transient single or double knockdown of flotillin-1 or caveolin-1 was not sufficient to increase cell migration over a short time course, suggesting complex crosstalk mechanisms. We propose a new model for CD44-dependent breast cancer cell migration, where CD44 must relocalize outside lipid rafts to drive cell migration. This could have implications for rafts as pharmacological targets to down-regulate cancer cell migration. PMID:23031255

Association of γ-Secretase with Lipid Rafts in Post-Golgi and Endosome Membranes*

PubMed Central

Vetrivel, Kulandaivelu S.; Cheng, Haipeng; Lin, William; Sakurai, Takashi; Li, Tong; Nukina, Nobuyuki; Wong, Philip C.; Xu, Huaxi; Thinakaran, Gopal

2005-01-01

Alzheimer’s disease-associated β-amyloid peptides (Aβ) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by β- and γ-secretases. There is growing evidence that cholesterol- and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major γ-secretase in neurons is a palmi-toylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the γ-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1−/−/PS2−/− and NCT−/− fibroblasts, γ-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires γ-secretase complex assembly. Biochemical evidence shows that subunits of the γ-secretase complex and three TGN/endosome-resident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1- or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of γ-secretase in lipid raft microdomains of post-Golgi and endosomes, organelles previously implicated in amyloidogenic processing of APP. PMID:15322084

Involvement of lipid rafts in adhesion-induced activation of Met and EGFR.

PubMed

Lu, Ying-Che; Chen, Hong-Chen

2011-10-27

Cell adhesion has been shown to induce activation of certain growth factor receptors in a ligand-independent manner. However, the mechanism for such activation remains obscure. Human epidermal carcinoma A431 cells were used as a model to examine the mechanism for adhesion-induced activation of hepatocyte growth factor receptor Met and epidermal growth factor receptor (EGFR). The cells were suspended and replated on culture dishes under various conditions. The phosphorylation of Met at Y1234/1235 and EGFR at Y1173 were used as indicators for their activation. The distribution of the receptors and lipid rafts on the plasma membrane were visualized by confocal fluorescent microscopy and total internal reflection microscopy. We demonstrate that Met and EGFR are constitutively activated in A431 cells, which confers proliferative and invasive potentials to the cells. The ligand-independent activation of Met and EGFR in A431 cells relies on cell adhesion to a substratum, but is independent of cell spreading, extracellular matrix proteins, and substratum stiffness. This adhesion-induced activation of Met and EGFR cannot be attributed to Src activation, production of reactive oxygen species, and the integrity of the cytoskeleton. In addition, we demonstrate that Met and EGFR are independently activated upon cell adhesion. However, partial depletion of Met and EGFR prevents their activation upon cell adhesion, suggesting that overexpression of the receptors is a prerequisite for their self-activation upon cell adhesion. Although Met and EGFR are largely distributed in 0.04% Triton-insoluble fractions (i.e. raft fraction), their activated forms are detected mainly in 0.04% Triton-soluble fractions (i.e. non-raft fraction). Upon cell adhesion, lipid rafts are accumulated at the cell surface close to the cell-substratum interface, while Met and EGFR are mostly excluded from the membrane enriched by lipid rafts. Our results suggest for the first time that cell adhesion to a

Triton promotes domain formation in lipid raft mixtures.

PubMed

Heerklotz, H

2002-11-01

Biological membranes are supposed to contain functional domains (lipid rafts) made up in particular of sphingomyelin and cholesterol, glycolipids, and certain proteins. It is often assumed that the application of the detergent Triton at 4 degrees C allows the isolation of these rafts as a detergent-resistant membrane fraction. The current study aims to clarify whether and how Triton changes the domain properties. To this end, temperature-dependent transitions in vesicles of an equimolar mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, egg sphingomyelin, and cholesterol were monitored at different Triton concentrations by differential scanning calorimetry and pressure perturbation calorimetry. Transitions initiated by the addition of Triton to the lipid mixture were studied by isothermal titration calorimetry, and the structure was investigated by (31)P-NMR. The results are discussed in terms of liquid-disordered (ld) and -ordered (lo) bilayer and micellar (mic) phases, and the typical sequence encountered with increasing Triton content or decreasing temperature is ld, ld + lo, ld + lo + mic, and lo + mic. That means that addition of Triton may create ordered domains in a homogeneous fluid membrane, which are, in turn, Triton resistant upon subsequent membrane solubilization. Hence, detergent-resistant membranes should not be assumed to resemble biological rafts in size, structure, composition, or even existence. Functional rafts may not be steady phenomena; they might form, grow, cluster or break up, shrink, and vanish according to functional requirements, regulated by rather subtle changes in the activity of membrane disordering or ordering compounds.

Triton promotes domain formation in lipid raft mixtures.

PubMed Central

Heerklotz, H

2002-01-01

Biological membranes are supposed to contain functional domains (lipid rafts) made up in particular of sphingomyelin and cholesterol, glycolipids, and certain proteins. It is often assumed that the application of the detergent Triton at 4 degrees C allows the isolation of these rafts as a detergent-resistant membrane fraction. The current study aims to clarify whether and how Triton changes the domain properties. To this end, temperature-dependent transitions in vesicles of an equimolar mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, egg sphingomyelin, and cholesterol were monitored at different Triton concentrations by differential scanning calorimetry and pressure perturbation calorimetry. Transitions initiated by the addition of Triton to the lipid mixture were studied by isothermal titration calorimetry, and the structure was investigated by (31)P-NMR. The results are discussed in terms of liquid-disordered (ld) and -ordered (lo) bilayer and micellar (mic) phases, and the typical sequence encountered with increasing Triton content or decreasing temperature is ld, ld + lo, ld + lo + mic, and lo + mic. That means that addition of Triton may create ordered domains in a homogeneous fluid membrane, which are, in turn, Triton resistant upon subsequent membrane solubilization. Hence, detergent-resistant membranes should not be assumed to resemble biological rafts in size, structure, composition, or even existence. Functional rafts may not be steady phenomena; they might form, grow, cluster or break up, shrink, and vanish according to functional requirements, regulated by rather subtle changes in the activity of membrane disordering or ordering compounds. PMID:12414701

Lenalidomide induces lipid raft assembly to enhance erythropoietin receptor signaling in myelodysplastic syndrome progenitors.

PubMed

McGraw, Kathy L; Basiorka, Ashley A; Johnson, Joseph O; Clark, Justine; Caceres, Gisela; Padron, Eric; Heaton, Ruth; Ozawa, Yukiyasu; Wei, Sheng; Sokol, Lubomir; List, Alan F

2014-01-01

Anemia remains the principal management challenge for patients with lower risk Myelodysplastic Syndromes (MDS). Despite appropriate cytokine production and cellular receptor display, erythropoietin receptor (EpoR) signaling is impaired. We reported that EpoR signaling is dependent upon receptor localization within lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity. Here, we show that MDS erythroid progenitors display markedly diminished raft assembly and smaller raft aggregates compared to normal controls (p = 0.005, raft number; p = 0.023, raft size). Because lenalidomide triggers raft coalescence in T-lymphocytes promoting immune synapse formation, we assessed effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lenalidomide treatment rapidly induced lipid raft formation accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase. The JAK2 phosphatase, CD45, a key negative regulator of EpoR signaling, was displaced from raft fractions. Lenalidomide treatment prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 and primary MDS erythroid progenitors, accompanied by increased STAT5 DNA binding in UT7 cells, and increased erythroid colony forming capacity in both UT7 and primary cells. Raft induction was associated with F-actin polymerization, which was blocked by Rho kinase inhibition. These data indicate that deficient raft integrity impairs EpoR signaling, and provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS.

Lenalidomide Induces Lipid Raft Assembly to Enhance Erythropoietin Receptor Signaling in Myelodysplastic Syndrome Progenitors

PubMed Central

McGraw, Kathy L.; Basiorka, Ashley A.; Johnson, Joseph O.; Clark, Justine; Caceres, Gisela; Padron, Eric; Heaton, Ruth; Ozawa, Yukiyasu; Wei, Sheng; Sokol, Lubomir; List, Alan F.

2014-01-01

Anemia remains the principal management challenge for patients with lower risk Myelodysplastic Syndromes (MDS). Despite appropriate cytokine production and cellular receptor display, erythropoietin receptor (EpoR) signaling is impaired. We reported that EpoR signaling is dependent upon receptor localization within lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity. Here, we show that MDS erythroid progenitors display markedly diminished raft assembly and smaller raft aggregates compared to normal controls (p = 0.005, raft number; p = 0.023, raft size). Because lenalidomide triggers raft coalescence in T-lymphocytes promoting immune synapse formation, we assessed effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lenalidomide treatment rapidly induced lipid raft formation accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase. The JAK2 phosphatase, CD45, a key negative regulator of EpoR signaling, was displaced from raft fractions. Lenalidomide treatment prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 and primary MDS erythroid progenitors, accompanied by increased STAT5 DNA binding in UT7 cells, and increased erythroid colony forming capacity in both UT7 and primary cells. Raft induction was associated with F-actin polymerization, which was blocked by Rho kinase inhibition. These data indicate that deficient raft integrity impairs EpoR signaling, and provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS. PMID:25469886

Lipid rafts mediate the interaction between myelin-associated glycoprotein (MAG) on myelin and MAG-receptors on neurons.

PubMed

Vinson, Mary; Rausch, Oliver; Maycox, Peter R; Prinjha, Rab K; Chapman, Debra; Morrow, Rachel; Harper, Alex J; Dingwall, Colin; Walsh, Frank S; Burbidge, Stephen A; Riddell, David R

2003-03-01

The interaction between myelin-associated glycoprotein (MAG), expressed at the periaxonal membrane of myelin, and receptors on neurons initiates a bidirectional signalling system that results in inhibition of neurite outgrowth and maintenance of myelin integrity. We show that this involves a lipid-raft to lipid-raft interaction on opposing cell membranes. MAG is exclusively located in low buoyancy Lubrol WX-insoluble membrane fractions isolated from whole brain, primary oligodendrocytes, or MAG-expressing CHO cells. Localisation within these domains is dependent on cellular cholesterol and occurs following terminal glycosylation in the trans-Golgi network, characteristics of association with lipid rafts. Furthermore, a recombinant form of MAG interacts specifically with lipid-raft fractions from whole brain and cultured cerebellar granule cells, containing functional MAG receptors GT1b and Nogo-66 receptor and molecules required for transduction of signal from MAG into neurons. The localisation of both MAG and MAG receptors within lipid rafts on the surface of opposing cells may create discrete areas of high avidity multivalent interaction, known to be critical for signalling into both cell types. Localisation within lipid rafts may provide a molecular environment that facilitates the interaction between MAG and multiple receptors and also between MAG ligands and molecules involved in signal transduction.

Dimerization controls the lipid raft partitioning of uPAR/CD87 and regulates its biological functions

PubMed Central

Cunningham, Orla; Andolfo, Annapaola; Santovito, Maria Lisa; Iuzzolino, Lucia; Blasi, Francesco; Sidenius, Nicolai

2003-01-01

The urokinase-type plasminogen activator receptor (uPAR/CD87) is a glycosylphosphatidylinositol-anchored membrane protein with multiple functions in extracellular proteolysis, cell adhesion, cell migration and proliferation. We now report that cell surface uPAR dimerizes and that dimeric uPAR partitions preferentially to detergent-resistant lipid rafts. Dimerization of uPAR did not require raft partitioning as the lowering of membrane cholesterol failed to reduce dimerization and as a transmembrane uPAR chimera, which does not partition to lipid rafts, also dimerized efficiently. While uPA bound to uPAR independently of its membrane localization and dimerization status, uPA-induced uPAR cleavage was strongly accelerated in lipid rafts. In contrast to uPA, the binding of Vn occurred preferentially to raft- associated dimeric uPAR and was completely blocked by cholesterol depletion. PMID:14609946

Hepatitis C Virus Induces the Localization of Lipid Rafts to Autophagosomes for Its RNA Replication

PubMed Central

Kim, Ja Yeon; Wang, Linya; Lee, Jiyoung

2017-01-01

ABSTRACT Autophagy plays important roles in maintaining cellular homeostasis. It uses double- or multiple-membrane vesicles termed autophagosomes to remove protein aggregates and damaged organelles from the cytoplasm for recycling. Hepatitis C virus (HCV) has been shown to induce autophagy to enhance its own replication. Here we describe a procedure that combines membrane flotation and affinity chromatography for the purification of autophagosomes from cells that harbor an HCV subgenomic RNA replicon. The purified autophagosomes had double- or multiple-membrane structures with a diameter ranging from 200 nm to 600 nm. The analysis of proteins associated with HCV-induced autophagosomes by proteomics led to the identification of HCV nonstructural proteins as well as proteins involved in membrane trafficking. Notably, caveolin-1, caveolin-2, and annexin A2, which are proteins associated with lipid rafts, were also identified. The association of lipid rafts with HCV-induced autophagosomes was confirmed by Western blotting, immunofluorescence microscopy, and immunoelectron microscopy. Their association with autophagosomes was also confirmed in HCV-infected cells. The association of lipid rafts with autophagosomes was specific to HCV, as it was not detected in autophagosomes induced by nutrient starvation. Further analysis indicated that the autophagosomes purified from HCV replicon cells could mediate HCV RNA replication in a lipid raft-dependent manner, as the depletion of cholesterol, a major component of lipid rafts, from autophagosomes abolished HCV RNA replication. Our studies thus demonstrated that HCV could specifically induce the association of lipid rafts with autophagosomes for its RNA replication. IMPORTANCE HCV can cause severe liver diseases, including cirrhosis and hepatocellular carcinoma, and is one of the most important human pathogens. Infection with HCV can lead to the reorganization of membrane structures in its host cells, including the induction of

Clathrin to Lipid Raft-Endocytosis via Controlled Surface Chemistry and Efficient Perinuclear Targeting of Nanoparticle.

PubMed

Chakraborty, Atanu; Jana, Nikhil R

2015-09-17

Nanoparticle interacts with live cells depending on their surface chemistry, enters into cell via endocytosis, and is commonly trafficked to an endosome/lysozome that restricts subcellular targeting options. Here we show that nanoparticle surface chemistry can be tuned to alter their cell uptake mechanism and subcellular trafficking. Quantum dot based nanoprobes of 20-30 nm hydrodynamic diameters have been synthesized with tunable surface charge (between +15 mV to -25 mV) and lipophilicity to influence their cellular uptake processes and subcellular trafficking. It is observed that cationic nanoprobe electrostatically interacts with cell membrane and enters into cell via clathrin-mediated endocytosis. At lower surface charge (between +10 mV to -10 mV), the electrostatic interaction with cell membrane becomes weaker, and additional lipid raft endocytosis is initiated. If a lipophilic functional group is introduced on a weakly anionic nanoparticle surface, the uptake mechanism shifts to predominant lipid raft-mediated endocytosis. In particular, the zwitterionic-lipophilic nanoprobe has the unique advantage as it weakly interacts with anionic cell membrane, migrates toward lipid rafts for interaction through lipophilic functional group, and induces lipid raft-mediated endocytosis. While predominate or partial clathrin-mediated entry traffics most of the nanoprobes to lysozome, predominate lipid raft-mediated entry traffics them to perinuclear region, particularly to the Golgi apparatus. This finding would guide in designing appropriate nanoprobe for subcellular targeting and delivery.

A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains[S

PubMed Central

Krager, Kimberly J.; Sarkar, Mitul; Twait, Erik C.; Lill, Nancy L.; Koland, John G.

2012-01-01

The submicroscopic spatial organization of cell surface receptors and plasma membrane signaling molecules is readily characterized by electron microscopy (EM) via immunogold labeling of plasma membrane sheets. Although various signaling molecules have been seen to segregate within plasma membrane microdomains, the biochemical identity of these microdomains and the factors affecting their formation are largely unknown. Lipid rafts are envisioned as submicron membrane subdomains of liquid ordered structure with differing lipid and protein constituents that define their specific varieties. To facilitate EM investigation of inner leaflet lipid rafts and the localization of membrane proteins therein, a unique genetically encoded reporter with the dually acylated raft-targeting motif of the Lck kinase was developed. This reporter, designated Lck-BAP-GFP, incorporates green fluorescent protein (GFP) and biotin acceptor peptide (BAP) modules, with the latter allowing its single-step labeling with streptavidin-gold. Lck-BAP-GFP was metabolically biotinylated in mammalian cells, distributed into low-density detergent-resistant membrane fractions, and was readily detected with avidin-based reagents. In EM images of plasma membrane sheets, the streptavidin-gold-labeled reporter was clustered in 20–50 nm microdomains, presumably representative of inner leaflet lipid rafts. The utility of the reporter was demonstrated in an investigation of the potential lipid raft localization of the epidermal growth factor receptor. PMID:22822037

Altered Dynamics of a Lipid Raft Associated Protein in a Kidney Model of Fabry Disease

PubMed Central

Labilloy, Anatália; Youker, Robert T.; Bruns, Jennifer R.; Kukic, Ira; Kiselyov, Kirill; Halfter, Willi; Finegold, David; do Monte, Semiramis Jamil Hadad; Weisz, Ora A.

2013-01-01

Accumulation of globotriaosylceramide (Gb3) and other neutral glycosphingolipids with galactosyl residues is the hallmark of Fabry disease, a lysosomal storage disorder caused by deficiency of the enzyme alpha-galactosidase A (α-gal A). These lipids are incorporated into the plasma membrane and intracellular membranes, with a preference for lipid rafts. Disruption of raft mediated cell processes is implicated in the pathogenesis of several human diseases, but little is known about the effects of the accumulation of glycosphingolipids on raft dynamics in the context of Fabry disease. Using siRNA technology, we have generated a polarized renal epithelial cell model of Fabry disease in Madin-Darby canine kidney cells. These cells present increased levels of Gb3 and enlarged lysosomes, and progressively accumulate zebra bodies. The polarized delivery of both raft-associated and raft-independent proteins was unaffected by α-gal A knockdown, suggesting that accumulation of Gb3 does not disrupt biosynthetic trafficking pathways. To assess the effect of α-gal A silencing on lipid raft dynamics, we employed number and brightness (N&B) analysis to measure the oligomeric status and mobility of the model glycosylphosphatidylinositol (GPI)-anchored protein GFP-GPI. We observed a significant increase in the oligomeric size of antibody-induced clusters of GFP-GPI at the plasma membrane of α-gal A silenced cells compared with control cells. Our results suggest that the interaction of GFP-GPI with lipid rafts may be altered in the presence of accumulated Gb3. The implications of our results with respect to the pathogenesis of Fabry disease are discussed. PMID:24215843

Identification of aquaporin-5 and lipid rafts in human resting saliva and their release into cevimeline-stimulated saliva.

PubMed

Pan, Yan; Iwata, Fusako; Wang, Di; Muraguchi, Masahiro; Ooga, Keiko; Ohmoto, Yasukazu; Takai, Masaaki; Cho, Gota; Kang, Jinsen; Shono, Masayuki; Li, Xue-jun; Okamura, Ko; Mori, Toyoki; Ishikawa, Yasuko

2009-01-01

It is unknown whether AQP5 and lipid rafts are released into human unstimulated (resting) saliva and saliva in response to secretagogues. In order to quantitate the salivary concentration of AQP5, we produced a polyclonal antibody for human AQP5 and developed an enzyme-like immunosorbent assay (ELISA). AQP5 and lipid rafts were identified in human resting saliva. The amount of AQP5 in resting saliva showed a diurnal variation with high levels during waking hours, and an age-related decrease in AQP5 was coincident with the volume of resting saliva. Cevimeline, a muscarinic acetylcholine receptor (mAChR) agonist, induced the release of AQP5 with lipid rafts, amylase, mucin, and lysozyme. Changes in saliva AQP5 levels after cevimeline administration occurred simultaneously with changes in saliva flow rates. Confocal microscopy revealed that AQP5 was located in the apical plasma membrane and showed a diffuse pattern in parotid glands under resting conditions. Following cevimeline administration, AQP5 was predominantly associated with the APM and was localized in the lumen. AQP5 and lipid rafts were released with salivary proteins from human salivary glands by the stimulation of M3 mAChRs, and that changes in saliva AQP5 levels can be used as an indicator of salivary flow rate and also as a useful index of M3 mAChR agonist's action on human salivary glands.

Lipid Rafts in Mast Cell Biology

PubMed Central

Silveira e Souza, Adriana Maria Mariano; Mazucato, Vivian Marino; Jamur, Maria Célia; Oliver, Constance

2011-01-01

Mast cells have long been recognized to have a direct and critical role in allergic and inflammatory reactions. In allergic diseases, these cells exert both local and systemic responses, including allergic rhinitis and anaphylaxis. Mast cell mediators are also related to many chronic inflammatory conditions. Besides the roles in pathological conditions, the biological functions of mast cells include roles in innate immunity, involvement in host defense mechanisms against parasites, immunomodulation of the immune system, tissue repair, and angiogenesis. Despite their growing significance in physiological and pathological conditions, much still remains to be learned about mast cell biology. This paper presents evidence that lipid rafts or raft components modulate many of the biological processes in mast cells, such as degranulation and endocytosis, play a role in mast cell development and recruitment, and contribute to the overall preservation of mast cell structure and organization. PMID:21490812

Juvenile-onset loss of lipid-raft domains in attractin-deficient mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Azouz, Abdallah; Gunn, Teresa M.; Duke-Cohan, Jonathan S.

2007-02-15

Mutations at the attractin (Atrn) locus in mice result in altered pigmentation on an agouti background, higher basal metabolic rate and juvenile-onset hypomyelination leading to neurodegeneration, while studies on human immune cells indicate a chemotaxis regulatory function. The underlying biochemical defect remains elusive. In this report we identify a role for attractin in plasma membrane maintenance. In attractin's absence there is a decline in plasma membrane glycolipid-enriched rafts from normal levels at 8 weeks to a complete absence by 24 weeks. The structural integrity of lipid rafts depends upon cholesterol and sphingomyelin, and can be identified by partitioning within ofmore » ganglioside GM{sub 1}. Despite a significant fall in cellular cholesterol with maturity, and a lesser fall in both membrane and total cellular GM{sub 1}, these parameters lag behind raft loss, and are normal when hypomyelination/neurodegeneration has already begun thus supporting consequence rather than cause. These findings can be recapitulated in Atrn-deficient cell lines propagated in vitro. Further, signal transduction through complex membrane receptor assemblies is not grossly disturbed despite the complete absence of lipid rafts. We find these results compatible with a role for attractin in plasma membrane maintenance and consistent with the proposal that the juvenile-onset hypomyelination and neurodegeneration represent a defect in attractin-mediated raft-dependent myelin biogenesis.« less

Lipid raft dynamics linked to sperm competency for fertilization in mice.

PubMed

Watanabe, Hitomi; Takeda, Rie; Hirota, Keiji; Kondoh, Gen

2017-05-01

It is well known that mammalian sperm acquires fertilization ability after several maturation processes, particularly within the female reproductive tract. In a previous study, we found that both glycosylphosphatidylinositol (GPI)-anchored protein (GPI-AP) release and lipid raft movement occur during the sperm maturation process. In several genetic studies, release of GPI-AP is a crucial step for sperm fertilization ability in the mouse. Here, we show that lipid raft movement is also fundamental for sperm to be competent for fertilization by comparing the sperm maturation process of two mouse inbred strains, C57BL/6 and BALB/c. We found that ganglioside GM1 movement was exclusively reduced in BALB/c compared with C57BL/6 among other examined sperm maturation parameters, such as GPI-AP release, sperm migration to the oviduct, cholesterol efflux, protein tyrosine phosphorylation and acrosome reaction, and was strongly linked to sperm fertility phenotype. The relationship between GM1 movement and in vitro fertilization ability was confirmed in other mouse strains, suggesting that lipid raft movement is one of the important steps for completing the sperm maturation process. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

"Lipid raft aging" in the human frontal cortex during nonpathological aging: gender influences and potential implications in Alzheimer's disease.

PubMed

Díaz, Mario; Fabelo, Noemí; Ferrer, Isidre; Marín, Raquel

2018-07-01

Lipid rafts are highly dynamic membrane domains featured by distinctive biochemical composition and physicochemical properties compared with the surrounding plasma membrane. These microstructures are associated not only with cellular signaling and communication in normal nerve cells but also with pathological processing of amyloid precursor protein in Alzheimer's disease. Using lipid rafts isolated from human frontal cortex in nondemented subjects aging 24 to 85 years, we demonstrate here that lipid structure of lipid rafts undergo significant alterations of specific lipid classes and phospholipid-bound fatty acids as brain cortex correlating with aging. Main changes affect levels of plasmalogens, polyunsaturated fatty acids (especially docosahexaenoic acid and arachidonic acid), total polar lipids (mainly phosphatidylinositol, sphingomyelin, sulfatides, and cerebrosides), and total neutral lipids (particularly cholesterol and sterol esters). Besides, relevant relationships between main fatty acids and/or lipid classes were altered in an age-related manner. This "lipid raft aging" exhibits clear gender differences and appear to be more pronounced in women than in men, especially in older (postmenopausal) women. The outcomes led us to conclude that human cortical lipid rafts are modified by aging in a gender-dependent fashion. Given the central role of bilayer lipid matrix in lipid rafts functionality and neuronal signaling, we hypothesize that these findings might underlie the higher prevalence of cognitive decline evolving toward Alzheimer's disease in postmenopausal women. Copyright © 2018 Elsevier Inc. All rights reserved.

Tumor exosomes induce tunneling nanotubes in lipid raft-enriched regions of human mesothelioma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thayanithy, Venugopal; Babatunde, Victor; Dickson, Elizabeth L.

Tunneling nanotubes (TnTs) are long, non-adherent, actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study, we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24–48 h; and this effect was most prominent in media conditions (low-serum, hyperglycemic medium) that support TnT formationmore » (1.3–1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs, in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs, which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation, and also lipid raft formation as a potential biomarker for TnT-forming cells. - Highlights: • Exosomes derived from malignant cells can stimulate an increased rate in the formation of tunneling nanotubes. • Tunneling nanotubes can serve as conduits for intercellular transfer of these exosomes. • Most notably, exosomes derived from benign mesothelial cells had no effect on nanotube formation. • Cells forming nanotubes were enriched in lipid rafts at a greater number compared with cells not forming nanotubes. • Our findings suggest causal and potentially synergistic association of exosomes

Localization of the Placental BCRP/ABCG2 Transporter to Lipid Rafts: Role for Cholesterol in Mediating Efflux Activity

PubMed Central

Szilagyi, John T.; Vetrano, Anna M.; Laskin, Jeffrey D.; Aleksunes, Lauren M.

2017-01-01

Introduction The breast cancer resistance protein (BCRP/ABCG2) is an efflux transporter in the placental barrier. By transporting chemicals from the fetal to the maternal circulation, BCRP limits fetal exposure to a range of drugs, toxicants, and endobiotics such as bile acids and hormones. The purpose of the present studies was to 1) determine whether BCRP localizes to highly-ordered, cholesterol-rich lipid raft microdomains in placenta microvillous membranes, and 2) determine the impact of cholesterol on BCRP-mediated placental transport in vitro. Methods BCRP expression was analyzed in lipid rafts isolated from placentas from healthy, term pregnancies and BeWo trophoblasts by density gradient ultracentrifugation. BeWo cells were also tested for their ability to efflux BCRP substrates after treatment with the cholesterol sequestrant methyl-β-cyclodextrin (MβCD, 5mM, 1 h) or the cholesterol synthesis inhibitor pravastatin (200μM, 48 h). Results and Discussion BCRP was found to co-localize with lipid raft proteins in detergent-resistant, lipid raft-containing fractions from placental microvillous membranes and BeWo cells. Treatment of BeWo cells with MβCD redistributed BCRP protein into higher density non-lipid raft fractions. Repletion of the cells with cholesterol restored BCRP localization to lipid raft-containing fractions. Treatment of BeWo cells with MβCD or pravastatin increased cellular retention of two BCRP substrates, the fluorescent dye Hoechst 33342 and the mycotoxin zearalenone. Repletion with cholesterol restored BCRP transporter activity. Taken together, these data demonstrate that cholesterol may play a critical role in the post-translational regulation of BCRP in placental lipid rafts. PMID:28623970

Localization of the placental BCRP/ABCG2 transporter to lipid rafts: Role for cholesterol in mediating efflux activity.

PubMed

Szilagyi, John T; Vetrano, Anna M; Laskin, Jeffrey D; Aleksunes, Lauren M

2017-07-01

The breast cancer resistance protein (BCRP/ABCG2) is an efflux transporter in the placental barrier. By transporting chemicals from the fetal to the maternal circulation, BCRP limits fetal exposure to a range of drugs, toxicants, and endobiotics such as bile acids and hormones. The purpose of the present studies was to 1) determine whether BCRP localizes to highly-ordered, cholesterol-rich lipid raft microdomains in placenta microvillous membranes, and 2) determine the impact of cholesterol on BCRP-mediated placental transport in vitro. BCRP expression was analyzed in lipid rafts isolated from placentas from healthy, term pregnancies and BeWo trophoblasts by density gradient ultracentrifugation. BeWo cells were also tested for their ability to efflux BCRP substrates after treatment with the cholesterol sequestrant methyl-β-cyclodextrin (MβCD, 5 mM, 1 h) or the cholesterol synthesis inhibitor pravastatin (200 μM, 48 h). BCRP was found to co-localize with lipid raft proteins in detergent-resistant, lipid raft-containing fractions from placental microvillous membranes and BeWo cells. Treatment of BeWo cells with MβCD redistributed BCRP protein into higher density non-lipid raft fractions. Repletion of the cells with cholesterol restored BCRP localization to lipid raft-containing fractions. Treatment of BeWo cells with MβCD or pravastatin increased cellular retention of two BCRP substrates, the fluorescent dye Hoechst 33342 and the mycotoxin zearalenone. Repletion with cholesterol restored BCRP transporter activity. Taken together, these data demonstrate that cholesterol may play a critical role in the post-translational regulation of BCRP in placental lipid rafts. Copyright © 2017 Elsevier Ltd. All rights reserved.

Xenon and Other Volatile Anesthetics Change Domain Structure in Model Lipid Raft Membranes

PubMed Central

Weinrich, Michael; Worcester, David L.

2014-01-01

Inhalation anesthetics have been in clinical use for over 160 years, but the molecular mechanisms of action continue to be investigated. Direct interactions with ion channels received much attention after it was found that anesthetics do not change the structure of homogeneous model membranes. However, it was recently found that halothane, a prototypical anesthetic, changes domain structure of a binary lipid membrane. The noble gas xenon is an excellent anesthetic and provides a pivotal test of the generality of this finding, extended to ternary lipid raft mixtures. We report that xenon and conventional anesthetics change the domain equilibrium in two canonical ternary lipid raft mixtures. These findings demonstrate a membrane-mediated mechanism whereby inhalation anesthetics can affect the lipid environment of trans-membrane proteins. PMID:24299622

Atomistic and coarse-grained computer simulations of raft-like lipid mixtures.

PubMed

Pandit, Sagar A; Scott, H Larry

2007-01-01

Computer modeling can provide insights into the existence, structure, size, and thermodynamic stability of localized raft-like regions in membranes. However, the challenges in the construction and simulation of accurate models of heterogeneous membranes are great. The primary obstacle in modeling the lateral organization within a membrane is the relatively slow lateral diffusion rate for lipid molecules. Microsecond or longer time-scales are needed to fully model the formation and stability of a raft in a membra ne. Atomistic simulations currently are not able to reach this scale, but they do provide quantitative information on the intermolecular forces and correlations that are involved in lateral organization. In this chapter, the steps needed to carry out and analyze atomistic simulations of hydrated lipid bilayers having heterogeneous composition are outlined. It is then shown how the data from a molecular dynamics simulation can be used to construct a coarse-grained model for the heterogeneous bilayer that can predict the lateral organization and stability of rafts at up to millisecond time-scales.

CD28 and lipid rafts coordinate recruitment of Lck to the immunological synapse of human T lymphocytes.

PubMed

Tavano, Regina; Gri, Giorgia; Molon, Barbara; Marinari, Barbara; Rudd, Christopher E; Tuosto, Loretta; Viola, Antonella

2004-11-01

In T lymphocytes, the Src family kinase Lck associates lipid rafts and accumulates at the immunological synapse (IS) during T cell stimulation by APCs. Using CD4- or CD28-deficient murine T cells, it was suggested that recruitment of Lck to the IS depends on CD4, whereas CD28 sustains Lck activation. However, in human resting T cells, CD28 is responsible for promoting recruitment of lipid rafts to the IS by an unknown mechanism. Thus, we performed a series of experiments to determine 1) whether Lck is recruited to the IS through lipid rafts; and 2) whether Lck recruitment to the IS of human resting T cells depends on CD4 or on CD28 engagement. We found that CD28, but not CD4, stimulation induced recruitment of Lck into detergent-resistant domains as well as its accumulation at the IS. We also found that Lck recruitment to the IS depends on the CD28 COOH-terminal PxxPP motif. Thus, the CD28-3A mutant, generated by substituting the prolines in positions 208, 211, and 212 with alanines, failed to induce Lck and lipid raft accumulation at the synapse. These results indicate that CD28 signaling orchestrates both Lck and lipid raft recruitment to the IS to amplify T cell activation.

7-Ketocholesterol Incorporation into Sphingolipid/Cholesterol-enriched (Lipid Raft) Domains Is Impaired by Vitamin E

PubMed Central

Royer, Marie-Charlotte; Lemaire-Ewing, Stéphanie; Desrumaux, Catherine; Monier, Serge; Pais de Barros, Jean-Paul; Athias, Anne; Néel, Dominique; Lagrost, Laurent

2009-01-01

Cholesterol oxides, in particular 7-ketocholesterol, are proatherogenic compounds that induce cell death in the vascular wall when localized in lipid raft domains of the cell membrane. Deleterious effects of 7-ketocholesterol can be prevented by vitamin E, but the molecular mechanism involved is unclear. In this study, unlike γ-tocopherol, the α-tocopherol vitamin E form was found to prevent 7-ketocholesterol-mediated apoptosis of A7R5 smooth muscle cells. To be operative, α-tocopherol needed to be added to the cells before 7-ketocholesterol, and its anti-apoptotic effect was reduced and even suppressed when added together or after 7-ketocholesterol, respectively. Both pre- and co-treatment of the cells with α-tocopherol resulted in the redistribution of 7-ketocholesterol out of the sphingolipid/cholesterol-enriched (lipid raft) domains. In turn, fewer amounts of α-tocopherol associated with lipid rafts on 7-ketocholesterol-pretreated cells compared with untreated cells, with no prevention of cell death in this case. In further support of the implication of lipid raft domains, the dephosphorylation/inactivation of Akt-PKB was involved in the 7-ketocholesterol-induced apoptosis. Akt-PKB dephosphorylation was prevented by α-tocopherol, but not γ-tocopherol pretreatment. PMID:19351882

Compartmentalization of endocannabinoids into lipid rafts in a microglial cell line devoid of caveorrlin-1

PubMed Central

Rimmerman, Neta; Bradshaw, Heather B; Kozela, Ewa; Levy, Rivka; Juknat, Ana; Vogel, Zvi

2012-01-01

BACKGROUND AND PURPOSE N-acyl ethanolamines (NAEs) and 2-arachidonoyl glycerol (2-AG) are endogenous cannabinoids and along with related lipids are synthesized on demand from membrane phospholipids. Here, we have studied the compartmentalization of NAEs and 2-AG into lipid raft fractions isolated from the caveolin-1-lacking microglial cell line BV-2, following vehicle or cannabidiol (CBD) treatment. Results were compared with those from the caveolin-1-positive F-11 cell line. EXPERIMENTAL APPROACH BV-2 cells were incubated with CBD or vehicle. Cells were fractionated using a detergent-free continuous OptiPrep density gradient. Lipids in fractions were quantified using HPLC/MS/MS. Proteins were measured using Western blot. KEY RESULTS BV-2 cells were devoid of caveolin-1. Lipid rafts were isolated from BV-2 cells as confirmed by co-localization with flotillin-1 and sphingomyelin. Small amounts of cannabinoid CB1 receptors were found in lipid raft fractions. After incubation with CBD, levels and distribution in lipid rafts of 2-AG, N-arachidonoyl ethanolamine (AEA), and N-oleoyl ethanolamine (OEA) were not changed. Conversely, the levels of the saturated N-stearoyl ethanolamine (SEA) and N-palmitoyl ethanolamine (PEA) were elevated in lipid raft fractions. In whole cells with growth medium, CBD treatment increased AEA and OEA time-dependently, while levels of 2-AG, PEA and SEA did not change. CONCLUSIONS AND IMPLICATIONS Whereas levels of 2-AG were not affected by CBD treatment, the distribution and levels of NAEs showed significant changes. Among the NAEs, the degree of acyl chain saturation predicted the compartmentalization after CBD treatment suggesting a shift in cell signalling activity. LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10

AFM of the ultrastructural and mechanical properties of lipid-raft-disrupted and/or cold-treated endothelial cells.

PubMed

Wu, Li; Huang, Jie; Yu, Xiaoxue; Zhou, Xiaoqing; Gan, Chaoye; Li, Ming; Chen, Yong

2014-02-01

The nonionic detergent extraction at 4 °C and the cholesterol-depletion-induced lipid raft disruption are the two widely used experimental strategies for lipid raft research. However, the effects of raft disruption and/or cold treatment on the ultrastructural and mechanical properties of cells are still unclear. Here, we evaluated the effects of raft disruption and/or cold (4 °C) treatment on these properties of living human umbilical vein endothelial cells (HUVECs). At first, the cholesterol-depletion-induced raft disruption was visualized by confocal microscopy and atomic force microscopy (AFM) in combination with fluorescent quantum dots. Next, the cold-induced cell contraction and the formation of end-branched filopodia were observed by confocal microscopy and AFM. Then, the cell-surface ultrastructures were imaged by AFM, and the data showed that raft disruption and cold treatment induced opposite effects on cell-surface roughness (a significant decrease and a significant increase, respectively). Moreover, the cell-surface mechanical properties (stiffness and adhesion force) of raft-disrupted- and/or cold-treated HUVECs were measured by the force measurement function of AFM. We found that raft disruption and cold treatment induced parallel effects on cell stiffness (increase) or adhesion force (decrease) and that the combination of the two treatments caused dramatically strengthened effects. Finally, raft disruption was found to significantly impair cell migration as previously reported, whereas temporary cold treatment only caused a slight but nonsignificant decrease in cell migration performed at physiological temperature. Although the mechanisms for causing these results might be complicated and more in-depth studies will be needed, our data may provide important information for better understanding the effects of raft disruption or cold treatment on cells and the two strategies for lipid raft research.

Antiproliferative effects of γ-tocotrienol are associated with lipid raft disruption in HER2-positive human breast cancer cells.

PubMed

Alawin, Osama A; Ahmed, Rayan A; Ibrahim, Baher A; Briski, Karen P; Sylvester, Paul W

2016-01-01

A large percentage of human breast cancers are characterized by excessive or aberrant HER2 activity. Lipid rafts are specialized microdomains within the plasma membrane that are required for HER2 activation and signal transduction. Since the anticancer activity of γ-tocotrienol is associated with suppression in HER2 signaling, studies were conducted to examine the effects of γ-tocotrienol on HER2 activation within the lipid raft microdomain in HER2-positive SKBR3 and BT474 human breast cancer cells. Treatment with 0-5μM γ-tocotrienol induced a significant dose-dependent inhibition in cancer cell growth after a 5-day culture period, and these growth inhibitory effects were associated with a reduction in HER2 dimerization and phosphorylation (activation). Phosphorylated HER2 was found to be primarily located in the lipid raft microdomain of the plasma membrane in vehicle-treated control groups, whereas γ-tocotrienol treatment significantly inhibited this effect. Assay of plasma membrane subcellular fractions showed that γ-tocotrienol also accumulates exclusively within the lipid raft microdomain. Hydroxypropyl-β-cyclodextrin (HPβCD) is an agent that disrupts lipid raft integrity. Acute exposure to 3mM HPβCD alone had no effect, whereas an acute 24-h exposure to 20μM γ-tocotrienol alone significantly decreased SKBR3 and BT474 cell viability. However, combined treatment with these agents greatly reduced γ-tocotrienol accumulation in the lipid raft microdomain and cytotoxicity. In summary, these findings demonstrate that the anticancer effects of γ-tocotrienol are associated with its accumulation in the lipid raft microdomain and subsequent interference with HER2 dimerization and activation in SKBR3 and BT474 human breast cancer cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Palmitoylation targets AKAP79 protein to lipid rafts and promotes its regulation of calcium-sensitive adenylyl cyclase type 8.

PubMed

Delint-Ramirez, Ilse; Willoughby, Debbie; Hammond, Gerald R V; Hammond, Gerald V R; Ayling, Laura J; Cooper, Dermot M F

2011-09-23

PKA anchoring proteins (AKAPs) optimize the efficiency of cAMP signaling by clustering interacting partners. Recently, AKAP79 has been reported to directly bind to adenylyl cyclase type 8 (AC8) and to regulate its responsiveness to store-operated Ca(2+) entry (SOCE). Although AKAP79 is well targeted to the plasma membrane via phospholipid associations with three N-terminal polybasic regions, recent studies suggest that AKAP79 also has the potential to be palmitoylated, which may specifically allow it to target the lipid rafts where AC8 resides and is regulated by SOCE. In this study, we have addressed the role of palmitoylation of AKAP79 using a combination of pharmacological, mutagenesis, and cell biological approaches. We reveal that AKAP79 is palmitoylated via two cysteines in its N-terminal region. This palmitoylation plays a key role in targeting the AKAP to lipid rafts in HEK-293 cells. Mutation of the two critical cysteines results in exclusion of AKAP79 from lipid rafts and alterations in its membrane diffusion behavior. This is accompanied by a loss of the ability of AKAP79 to regulate SOCE-dependent AC8 activity in intact cells and decreased PKA-dependent phosphorylation of raft proteins, including AC8. We conclude that palmitoylation plays a key role in the targeting and action of AKAP79. This novel property of AKAP79 adds an unexpected regulatory and targeting option for AKAPs, which may be exploited in the cellular context.

Lipid rafts regulate PCB153-induced disruption of occludin and brain endothelial barrier function through protein phosphatase 2A and matrix metalloproteinase-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eum, Sung Yong, E-mail: seum@miami.edu; Jaraki, Dima; András, Ibolya E.

Occludin is an essential integral transmembrane protein regulating tight junction (TJ) integrity in brain endothelial cells. Phosphorylation of occludin is associated with its localization to TJ sites and incorporation into intact TJ assembly. The present study is focused on the role of lipid rafts in polychlorinated biphenyl (PCB)-induced disruption of occludin and endothelial barrier function. Exposure of human brain endothelial cells to 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) induced dephosphorylation of threonine residues of occludin and displacement of occludin from detergent-resistant membrane (DRM)/lipid raft fractions within 1 h. Moreover, lipid rafts modulated the reduction of occludin level through activation of matrix metalloproteinase 2 (MMP-2)more » after 24 h PCB153 treatment. Inhibition of protein phosphatase 2A (PP2A) activity by okadaic acid or fostriecin markedly protected against PCB153-induced displacement of occludin and increased permeability of endothelial cells. The implication of lipid rafts and PP2A signaling in these processes was further defined by co-immunoprecipitation of occludin with PP2A and caveolin-1, a marker protein of lipid rafts. Indeed, a significant MMP-2 activity was observed in lipid rafts and was increased by exposure to PCB153. The pretreatment of MMP-2 inhibitors protected against PCB153-induced loss of occludin and disruption of lipid raft structure prevented the increase of endothelial permeability. Overall, these results indicate that lipid raft-associated processes, such as PP2A and MMP-2 activation, participate in PCB153-induced disruption of occludin function in brain endothelial barrier. This study contributes to a better understanding of the mechanisms leading to brain endothelial barrier dysfunction in response to exposure to environmental pollutants, such as ortho-substituted PCBs. - Highlights: • PCB153 disturbed human brain endothelial barrier through disruption of occludin. • Lipid raft

Hybrid and Nonhybrid Lipids Exert Common Effects on Membrane Raft Size and Morphology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heberle, Frederick A; Doktorova, Milka; Goh, Shih Lin

2013-01-01

Nanometer-scale domains in cholesterolrich model membranes emulate lipid rafts in cell plasma membranes (PMs). The physicochemical mechanisms that maintain a finite, small domain size are, however, not well understood. A special role has been postulated for chainasymmetric or hybrid lipids having a saturated sn-1 chain and an unsaturated sn-2 chain. Hybrid lipids generate nanodomains in some model membranes and are also abundant in the PM. It was proposed that they align in a preferred orientation at the boundary of ordered and disordered phases, lowering the interfacial energy and thus reducing domain size. We used small-angle neutron scattering and fluorescence techniquesmore » to detect nanoscopic and modulated liquid phase domains in a mixture composed entirely of nonhybrid lipids and cholesterol. Our results are indistinguishable from those obtained previously for mixtures containing hybrid lipids, conclusively showing that hybrid lipids are not required for the formation of nanoscopic liquid domains and strongly implying a common mechanism for the overall control of raft size and morphology. We discuss implications of these findings for theoretical descriptions of nanodomains.« less

Titanium particles activate Toll-like Receptor 4 independently of lipid rafts in RAW264.7 murine macrophages

PubMed Central

Islam, Andrew S.; Beidelschies, Michelle A.; Huml, Anne; Greenfield, Edward M.

2010-01-01

Adherent PAMPs (pathogen-associated molecular patterns) act through Toll-like receptor2 (TLR2) and TLR4 to increase the biological activity of orthopaedic wear particles in cell culture and animal models of implant loosening. This study tested whether this is dependent on TLR association with lipid rafts as reported for the response to soluble TLR ligands. For this purpose, RAW264.7 murine macrophages were activated by exposure to titanium particles with adherent PAMPs, soluble lipopolysaccharide (LPS), soluble lipotecichoic acid (LTA), or heat-killed bacteria that had been extensively washed to remove soluble PAMPs. Lipid rafts were isolated by two independent methods and the location of TLR4 and TLR2 was analyzed by western blotting. The cognate TLRs associated with lipid rafts when the macrophages were activated with soluble LPS and LTA but not after stimulation with either titanium particles with adherent PAMPs or heat-killed bacteria. The lipid raft disruptor, methyl-β-cyclodextrin, dose-dependently inhibited TNFα release in response to LPS but had no affect on TNFα release in response to titanium particles with adherent PAMPs. We conclude, therefore, that titanium particles with adherent PAMPs and heat-killed bacteria activate TLR2 and TLR4 in macrophages without inducing either TLR to associate with lipid rafts. These results have important implications for the mechanisms of orthopaedic implant loosening as well the mechanisms for TLR activation in other inflammatory situations. PMID:20806319

Clustering T cell GM1 Lipid Rafts Increases Cellular Resistance to Shear on Fibronectin through Changes in Integrin Affinity and Cytoskeletal Dynamics

PubMed Central

Mitchell, Jason S.; Brown, Wells S.; Woodside, Darren G.; Vanderslice, Peter; McIntyre, Bradley W.

2008-01-01

Lipid rafts are small laterally mobile microdomains that are highly enriched in lymphocyte signaling molecules. GM1 gangliosides are a common lipid raft component and have been shown to be important in many T cell functions. The aggregation of specific GM1 lipid rafts can control many T cell activation events, including their novel association with T cell integrins. We found that clustering GM1 lipid rafts can regulate β1 integrin function. This was apparent through increased resistance to shear flow dependent detachment of T cells adherent to the α4β1 and α5β1 integrin ligand fibronectin (FN). Adhesion strengthening as a result of clustering GM1 enriched lipid rafts correlated with increased cellular rigidity and morphology through the localization of cortical F-actin, the resistance to shear induced cell stretching, and an increase in the surface area and symmetry of the contact area between the cell surface and adhesive substrate. Furthermore, clustering GM1 lipid rafts could initiate integrin “inside-out” signaling mechanisms. This was seen through increased integrin-cytoskeleton associations and enhanced soluble binding of FN and VCAM-1 suggesting the induction of high affinity integrin conformations. The activation of these adhesion strengthening characteristics appear to be specific for the aggregation of GM1 lipid rafts as the aggregation of the heterogeneous raft associated molecule CD59 failed to activate these functions. These findings indicate a novel mechanism to signal to β1 integrins and to activate adhesion strengthening processes. PMID:19139760

A novel mechanism of regulating breast cancer cell migration via palmitoylation-dependent alterations in the lipid raft affiliation of CD44

PubMed Central

2014-01-01

Introduction Most breast cancer-related deaths result from metastasis, a process involving dynamic regulation of tumour cell adhesion and migration. The adhesion protein CD44, a key regulator of cell migration, is enriched in cholesterol-enriched membrane microdomains termed lipid rafts. We recently reported that raft affiliation of CD44 negatively regulates interactions with its migratory binding partner ezrin. Since raft affiliation is regulated by post-translational modifications including palmitoylation, we sought to establish the contribution of CD44 palmitoylation and lipid raft affiliation to cell migration. Methods Recovery of CD44 and its binding partners from raft versus non-raft membrane microdomains was profiled in non-migrating and migrating breast cancer cell lines. Site-directed mutagenesis was used to introduce single or double point mutations into both CD44 palmitoylation sites (Cys286 and Cys295), whereupon the implications for lipid raft recovery, phenotype, ezrin co-precipitation and migratory behaviour was assessed. Finally CD44 palmitoylation status and lipid raft affiliation was assessed in primary cultures from a small panel of breast cancer patients. Results CD44 raft affiliation was increased during migration of non-invasive breast cell lines, but decreased during migration of highly-invasive breast cells. The latter was paralleled by increased CD44 recovery in non-raft fractions, and exclusive non-raft recovery of its binding partners. Point mutation of CD44 palmitoylation sites reduced CD44 raft affiliation in invasive MDA-MB-231 cells, increased CD44-ezrin co-precipitation and accordingly enhanced cell migration. Expression of palmitoylation-impaired (raft-excluded) CD44 mutants in non-invasive MCF-10a cells was sufficient to reversibly induce the phenotypic appearance of epithelial-to-mesenchymal transition and to increase cell motility. Interestingly, cell migration was associated with temporal reductions in CD44 palmitoylation in

A novel mechanism of regulating breast cancer cell migration via palmitoylation-dependent alterations in the lipid raft affiliation of CD44.

PubMed

Babina, Irina S; McSherry, Elaine A; Donatello, Simona; Hill, Arnold D K; Hopkins, Ann M

2014-02-10

Most breast cancer-related deaths result from metastasis, a process involving dynamic regulation of tumour cell adhesion and migration. The adhesion protein CD44, a key regulator of cell migration, is enriched in cholesterol-enriched membrane microdomains termed lipid rafts. We recently reported that raft affiliation of CD44 negatively regulates interactions with its migratory binding partner ezrin. Since raft affiliation is regulated by post-translational modifications including palmitoylation, we sought to establish the contribution of CD44 palmitoylation and lipid raft affiliation to cell migration. Recovery of CD44 and its binding partners from raft versus non-raft membrane microdomains was profiled in non-migrating and migrating breast cancer cell lines. Site-directed mutagenesis was used to introduce single or double point mutations into both CD44 palmitoylation sites (Cys286 and Cys295), whereupon the implications for lipid raft recovery, phenotype, ezrin co-precipitation and migratory behaviour was assessed. Finally CD44 palmitoylation status and lipid raft affiliation was assessed in primary cultures from a small panel of breast cancer patients. CD44 raft affiliation was increased during migration of non-invasive breast cell lines, but decreased during migration of highly-invasive breast cells. The latter was paralleled by increased CD44 recovery in non-raft fractions, and exclusive non-raft recovery of its binding partners. Point mutation of CD44 palmitoylation sites reduced CD44 raft affiliation in invasive MDA-MB-231 cells, increased CD44-ezrin co-precipitation and accordingly enhanced cell migration. Expression of palmitoylation-impaired (raft-excluded) CD44 mutants in non-invasive MCF-10a cells was sufficient to reversibly induce the phenotypic appearance of epithelial-to-mesenchymal transition and to increase cell motility. Interestingly, cell migration was associated with temporal reductions in CD44 palmitoylation in wild-type breast cells. Finally

Analysis of the interaction between respiratory syncytial virus and lipid-rafts in Hep2 cells during infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, Gaie; Jeffree, Chris E.; McDonald, Terence

2004-10-01

The assembly of respiratory syncytial virus (RSV) in lipid-rafts was examined in Hep2 cells. Confocal and electron microscopy showed that during RSV assembly, the cellular distribution of the complement regulatory proteins, decay accelerating factor (CD55) and CD59, changes and high levels of these cellular proteins are incorporated into mature virus filaments. The detergent-solubility properties of CD55, CD59, and the RSV fusion (F) protein were found to be consistent with each protein being located predominantly within lipid-raft structures. The levels of these proteins in cell-released virus were examined by immunoelectronmicroscopy and found to account for between 5% and 15% of themore » virus attachment (G) glycoprotein levels. Collectively, our findings suggest that an intimate association exists between RSV and lipid-raft membranes and that significant levels of these host-derived raft proteins, such as those regulating complement activation, are subsequently incorporated into the envelope of mature virus particles.« less

Keratin impact on PKCδ- and ASMase-mediated regulation of hepatocyte lipid raft size - implication for FasR-associated apoptosis.

PubMed

Gilbert, Stéphane; Loranger, Anne; Omary, M Bishr; Marceau, Normand

2016-09-01

Keratins are epithelial cell intermediate filament (IF) proteins that are expressed as pairs in a cell-differentiation-regulated manner. Hepatocytes express the keratin 8 and 18 pair (denoted K8/K18) of IFs, and a loss of K8 or K18, as in K8-null mice, leads to degradation of the keratin partner. We have previously reported that a K8/K18 loss in hepatocytes leads to altered cell surface lipid raft distribution and more efficient Fas receptor (FasR, also known as TNFRSF6)-mediated apoptosis. We demonstrate here that the absence of K8 or transgenic expression of the K8 G62C mutant in mouse hepatocytes reduces lipid raft size. Mechanistically, we find that the lipid raft size is dependent on acid sphingomyelinase (ASMase, also known as SMPD1) enzyme activity, which is reduced in absence of K8/K18. Notably, the reduction of ASMase activity appears to be caused by a less efficient redistribution of surface membrane PKCδ toward lysosomes. Moreover, we delineate the lipid raft volume range that is required for an optimal FasR-mediated apoptosis. Hence, K8/K18-dependent PKCδ- and ASMase-mediated modulation of lipid raft size can explain the more prominent FasR-mediated signaling resulting from K8/K18 loss. The fine-tuning of ASMase-mediated regulation of lipid rafts might provide a therapeutic target for death-receptor-related liver diseases. © 2016. Published by The Company of Biologists Ltd.

Lipid Raft-dependent Glucagon-like Peptide-2 Receptor Trafficking Occurs Independently of Agonist-induced Desensitization

PubMed Central

Estall, Jennifer L.; Yusta, Bernardo; Drucker, Daniel J.

2004-01-01

The intestinotrophic and cytoprotective actions of glucagon-like peptide-2 (GLP-2) are mediated by the GLP-2 receptor (GLP-2R), a member of the class II glucagon-secretin G protein-coupled receptor superfamily. Although native GLP-2 exhibits a short circulating half-life, long-acting degradation-resistant GLP-2 analogues are being evaluated for therapeutic use in human subjects. Accordingly, we examined the mechanisms regulating signaling, internalization, and trafficking of the GLP-2R to identify determinants of receptor activation and desensitization. Heterologous cells expressing the transfected rat or human GLP-2R exhibited a rapid, dose-dependent, and prolonged desensitization of the GLP-2–stimulated cAMP response and a sustained GLP-2–induced decrease in levels of cell surface receptor. Surprisingly, inhibitors of clathrin-dependent endocytosis failed to significantly decrease GLP-2R internalization, whereas cholesterol sequestration inhibited ligand-induced receptor internalization and potentiated homologous desensitization. The hGLP-2R localized to both Triton X-100–soluble and –insoluble (lipid raft) cellular fractions and colocalized transiently with the lipid raft marker caveolin-1. Although GLP-2R endocytosis was dependent on lipid raft integrity, the receptor transiently associated with green fluorescent protein tagged-early endosome antigen 1–positive vesicles and inhibitors of endosomal acidification attenuated the reappearance of the GLP-2R on the cell surface. Our data demonstrate that GLP-2R desensitization and raft-dependent trafficking represent distinct and independent cellular mechanisms and provide new evidence implicating the importance of a clathrin- and dynamin-independent, lipid raft-dependent pathway for homologous G protein-coupled receptor internalization. PMID:15169869

Keratin impact on PKCδ- and ASMase-mediated regulation of hepatocyte lipid raft size – implication for FasR-associated apoptosis

PubMed Central

Gilbert, Stéphane; Loranger, Anne; Omary, M. Bishr

2016-01-01

ABSTRACT Keratins are epithelial cell intermediate filament (IF) proteins that are expressed as pairs in a cell-differentiation-regulated manner. Hepatocytes express the keratin 8 and 18 pair (denoted K8/K18) of IFs, and a loss of K8 or K18, as in K8-null mice, leads to degradation of the keratin partner. We have previously reported that a K8/K18 loss in hepatocytes leads to altered cell surface lipid raft distribution and more efficient Fas receptor (FasR, also known as TNFRSF6)-mediated apoptosis. We demonstrate here that the absence of K8 or transgenic expression of the K8 G62C mutant in mouse hepatocytes reduces lipid raft size. Mechanistically, we find that the lipid raft size is dependent on acid sphingomyelinase (ASMase, also known as SMPD1) enzyme activity, which is reduced in absence of K8/K18. Notably, the reduction of ASMase activity appears to be caused by a less efficient redistribution of surface membrane PKCδ toward lysosomes. Moreover, we delineate the lipid raft volume range that is required for an optimal FasR-mediated apoptosis. Hence, K8/K18-dependent PKCδ- and ASMase-mediated modulation of lipid raft size can explain the more prominent FasR-mediated signaling resulting from K8/K18 loss. The fine-tuning of ASMase-mediated regulation of lipid rafts might provide a therapeutic target for death-receptor-related liver diseases. PMID:27422101

Impact of lipid rafts on the T -cell-receptor and peptide-major-histocompatibility-complex interactions under different measurement conditions

NASA Astrophysics Data System (ADS)

Li, Long; Xu, Guang-Kui; Song, Fan

2017-01-01

The interactions between T-cell receptor (TCR) and peptide-major-histocompatibility complex (pMHC), which enable T-cell development and initiate adaptive immune responses, have been intensively studied. However, a central issue of how lipid rafts affect the TCR-pMHC interactions remains unclear. Here, by using a statistical-mechanical membrane model, we show that the binding affinity of TCR and pMHC anchored on two apposing cell membranes is significantly enhanced because of the lipid raft-induced signaling protein aggregation. This finding may provide an alternative insight into the mechanism of T-cell activation triggered by very low densities of pMHC. In the case of cell-substrate adhesion, our results indicate that the loss of lateral mobility of the proteins on the solid substrate leads to the inhibitory effect of lipid rafts on TCR-pMHC interactions. Our findings help to understand why different experimental methods for measuring the impact of lipid rafts on the receptor-ligand interactions have led to contradictory conclusions.

Membrane raft association is a determinant of plasma membrane localization.

PubMed

Diaz-Rohrer, Blanca B; Levental, Kandice R; Simons, Kai; Levental, Ilya

2014-06-10

The lipid raft hypothesis proposes lateral domains driven by preferential interactions between sterols, sphingolipids, and specific proteins as a central mechanism for the regulation of membrane structure and function; however, experimental limitations in defining raft composition and properties have prevented unequivocal demonstration of their functional relevance. Here, we establish a quantitative, functional relationship between raft association and subcellular protein sorting. By systematic mutation of the transmembrane and juxtamembrane domains of a model transmembrane protein, linker for activation of T-cells (LAT), we generated a panel of variants possessing a range of raft affinities. These mutations revealed palmitoylation, transmembrane domain length, and transmembrane sequence to be critical determinants of membrane raft association. Moreover, plasma membrane (PM) localization was strictly dependent on raft partitioning across the entire panel of unrelated mutants, suggesting that raft association is necessary and sufficient for PM sorting of LAT. Abrogation of raft partitioning led to mistargeting to late endosomes/lysosomes because of a failure to recycle from early endosomes. These findings identify structural determinants of raft association and validate lipid-driven domain formation as a mechanism for endosomal protein sorting.

Synaptic membrane rafts: traffic lights for local neurotrophin signaling?

PubMed

Zonta, Barbara; Minichiello, Liliana

2013-10-18

Lipid rafts, cholesterol and lipid rich microdomains, are believed to play important roles as platforms for the partitioning of transmembrane and synaptic proteins involved in synaptic signaling, plasticity, and maintenance. There is increasing evidence of a physical interaction between post-synaptic densities and post-synaptic lipid rafts. Localization of proteins within lipid rafts is highly regulated, and therefore lipid rafts may function as traffic lights modulating and fine-tuning neuronal signaling. The tyrosine kinase neurotrophin receptors (Trk) and the low-affinity p75 neurotrophin receptor (p75(NTR)) are enriched in neuronal lipid rafts together with the intermediates of downstream signaling pathways, suggesting a possible role of rafts in neurotrophin signaling. Moreover, neurotrophins and their receptors are involved in the regulation of cholesterol metabolism. Cholesterol is an important component of lipid rafts and its depletion leads to gradual loss of synapses, underscoring the importance of lipid rafts for proper neuronal function. Here, we review and discuss the idea that translocation of neurotrophin receptors in synaptic rafts may account for the selectivity of their transduced signals.

Rafts, Nanoparticles and Neural Disease

PubMed Central

Gulati, Vishal; Wallace, Ron

2012-01-01

This review examines the role of membrane rafts in neural disease as a rationale for drug targeting utilizing lipid-based nanoparticles. The article begins with an overview of methodological issues involving the existence, sizes, and lifetimes of rafts, and then examines raft function in the etiologies of three major neural diseases—epilepsy, Parkinson’s disease, and Alzheimer’s disease—selected as promising candidates for raft-based therapeutics. Raft-targeting drug delivery systems involving liposomes and solid lipid nanoparticles are then examined in detail. PMID:28348305

Carbachol-mediated endocytosis of NHE3 involves a clathrin-independent mechanism requiring lipid rafts and Cdc42.

PubMed

Zachos, Nicholas C; Alamelumangpuram, Bharath; Lee, Luke J; Wang, Peng; Kovbasnjuk, Olga

2014-01-01

In intestinal epithelial cells, acute regulation of the brush border Na(+)/H(+) exchanger, NHE3, usually occurs by changes in endocytosis and/or exocytosis. Constitutive NHE3 endocytosis involves clathrin. Carbachol (CCH), which elevates intracellular Ca(2+) ([Ca(2+)]i), decreases NHE3 activity and stimulates endocytosis; however, the mechanism involved in calcium-mediated endocytosis of NHE3 is unclear. A pool of NHE3 resides in lipid rafts, which contributes to basal, but not cAMP-mediated, NHE3 trafficking, suggesting that an alternative mechanism exists for NHE3 endocytosis. Cdc42 was demonstrated to play an integral role in some cases of cholesterol-sensitive, clathrin-independent endocytosis. Therefore, the current study was designed to test the hypotheses that (1) clathrin-mediated endocytosis (CME) is involved in constitutive, but not CCH-mediated, endocytosis of NHE3, and (2) CCH-mediated endocytosis of NHE3 occurs through a lipid raft, activated Cdc42-dependent pathway that does not involve clathrin. The role of Cdc42 and lipid rafts on NHE3 activity and endocytosis were investigated in polarized Caco-2/BBe cells using pharmacological and shRNA knockdown approaches. Basal NHE3 activity was increased in the presence of CME blockers (chlorpromazine; K(+) depletion) supporting previous reports that constitutive NHE3 endocytosis is clathrin dependent. In contrast, CCH-inhibition of NHE3 activity was abolished in Caco-2/BBe cells treated with MβCD (to disrupt lipid rafts) as well as in Cdc42 knockdown cells but was unaffected by CME blockers. CCH-mediated inhibition of NHE3 activity is not dependent on clathrin and involves lipid rafts and requires Cdc42. © 2014 S. Karger AG, Basel.

Carbachol-Mediated Endocytosis of NHE3 Involves a Clathrin-Independent Mechanism Requiring Lipid Rafts and Cdc42

PubMed Central

Zachos, Nicholas C.; Alamelumangpuram, Bharath; Lee, Luke J.; Wang, Peng; Kovbasnjuk, Olga

2014-01-01

Background In intestinal epithelial cells, acute regulation of the brush border Na+/H+ exchanger, NHE3, usually occurs by changes in endocytosis and/or exocytosis. Constitutive NHE3 endocytosis involves clathrin. Carbachol (CCH), which elevates intracellular Ca2+ ([Ca2+]i), decreases NHE3 activity and stimulates endocytosis; however, the mechanism involved in calcium-mediated endocytosis of NHE3 is unclear. A pool of NHE3 resides in lipid rafts, which contributes to basal, but not cAMP-mediated, NHE3 trafficking, suggesting that an alternative mechanism exists for NHE3 endocytosis. Cdc42 was demonstrated to play an integral role in some cases of cholesterol-sensitive, clathrin-independent endocytosis. Therefore, the current study was designed to test the hypotheses that (1) clathrin-mediated endocytosis (CME) is involved in constitutive, but not CCH-mediated, endocytosis of NHE3, and (2) CCH-mediated endocytosis of NHE3 occurs through a lipid raft, activated Cdc42-dependent pathway that does not involve clathrin. Methods The role of Cdc42 and lipid rafts on NHE3 activity and endocytosis were investigated in polarized Caco-2/BBe cells using pharmacological and shRNA knockdown approaches. Results Basal NHE3 activity was increased in the presence of CME blockers (chlorpromazine; K+ depletion) supporting previous reports that constitutive NHE3 endocytosis is clathrin dependent. In contrast, CCH-inhibition of NHE3 activity was abolished in Caco-2/BBe cells treated with MβCD (to disrupt lipid rafts) as well as in Cdc42 knockdown cells but was unaffected by CME blockers. Conclusion CCH-mediated inhibition of NHE3 activity is not dependent on clathrin and involves lipid rafts and requires Cdc42. PMID:24713550

Lipid Domain Structure of the Plasma Membrane Revealed by Patching of Membrane Components

PubMed Central

Harder, Thomas; Scheiffele, Peter; Verkade, Paul; Simons, Kai

1998-01-01

Lateral assemblies of glycolipids and cholesterol, “rafts,” have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T–lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components. PMID:9585412

Motility and stem cell properties induced by the epithelial-mesenchymal transition require destabilization of lipid rafts

PubMed Central

Prijic, Sara; Chen, Xiaoling; Levental, Ilya; Chang, Jeffrey T.

2016-01-01

The Epithelial-Mesenchymal Transition (EMT) is a developmental program that provides cancer cells with the characteristics necessary for metastasis, including increased motility and stem cell properties. The cellular and molecular mechanisms underlying this process are not yet fully understood, hampering efforts to develop therapeutics. In recent years, it has become apparent that EMT is accompanied by wholesale changes in diverse signaling pathways that are initiated by proteins at the plasma membrane (PM). The PM contains thousands of lipid and protein species that are dynamically and spatially organized into lateral membrane domains, an example of which are lipid rafts. Since one of the major functions of rafts is modulation of signaling originating at the PM, we hypothesized that the signaling changes occurring during an EMT are associated with alterations in PM organization. To test this hypothesis, we used Giant Plasma Membrane Vesicles (GPMVs) to study the organization of intact plasma membranes isolated from live cells. We observed that induction of EMT significantly destabilized lipid raft domains. Further, this reduction in stability was crucial for the maintenance of the stem cell phenotype and EMT-induced remodeling of PM-orchestrated pathways. Exogenously increasing raft stability by feeding cells with ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) repressed these phenotypes without altering EMT markers, and inhibited the metastatic capacity of breast cancer cells. Hence, modulating raft properties regulates cell phenotype, suggesting a novel approach for targeting the impact of EMT in cancer. PMID:27303921

Lipid rafts are required for signal transduction by angiotensin II receptor type 1 in neonatal glomerular mesangial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adebiyi, Adebowale, E-mail: aadebiyi@uthsc.edu; Soni, Hitesh; John, Theresa A.

Angiotensin II (ANG-II) receptors (AGTRs) contribute to renal physiology and pathophysiology, but the underlying mechanisms that regulate AGTR function in glomerular mesangium are poorly understood. Here, we show that AGTR1 is the functional AGTR subtype expressed in neonatal pig glomerular mesangial cells (GMCs). Cyclodextrin (CDX)-mediated cholesterol depletion attenuated cell surface AGTR1 protein expression and ANG-II-induced intracellular Ca{sup 2+} ([Ca{sup 2+}]{sub i}) elevation in the cells. The COOH-terminus of porcine AGTR1 contains a caveolin (CAV)-binding motif. However, neonatal GMCs express CAV-1, but not CAV-2 and CAV-3. Colocalization and in situ proximity ligation assay detected an association between endogenous AGTR1 and CAV-1more » in the cells. A synthetic peptide corresponding to the CAV-1 scaffolding domain (CSD) sequence also reduced ANG-II-induced [Ca{sup 2+}]{sub i} elevation in the cells. Real-time imaging of cell growth revealed that ANG-II stimulates neonatal GMC proliferation. ANG-II-induced GMC growth was attenuated by EMD 66684, an AGTR1 antagonist; BAPTA, a [Ca{sup 2+}]{sub i} chelator; KN-93, a Ca{sup 2+}/calmodulin-dependent protein kinase II inhibitor; CDX; and a CSD peptide, but not PD 123319, a selective AGTR2 antagonist. Collectively, our data demonstrate [Ca{sup 2+}]{sub i}-dependent proliferative effect of ANG-II and highlight a critical role for lipid raft microdomains in AGTR1-mediated signal transduction in neonatal GMCs. - Highlights: • AGTR1 is the functional AGTR subtype expressed in neonatal mesangial cells. • Endogenous AGTR1 associates with CAV-1 in neonatal mesangial cells. • Lipid raft disruption attenuates cell surface AGTR1 protein expression. • Lipid raft disruption reduces ANG-II-induced [Ca{sup 2+}]{sub i} elevation in neonatal mesangial cells. • Lipid raft disruption inhibits ANG-II-induced neonatal mesangial cell growth.« less

A Mechanistic Model of Early FcεRI Signaling: Lipid Rafts and the Question of Protection from Dephosphorylation

PubMed Central

Barua, Dipak; Goldstein, Byron

2012-01-01

We present a model of the early events in mast cell signaling mediated by FcεRI where the plasma membrane is composed of many small ordered lipid domains (rafts), surrounded by a non-order region of lipids consisting of the remaining plasma membrane. The model treats the rafts as transient structures that constantly form and breakup, but that maintain a fixed average number per cell. The rafts have a high propensity for harboring Lyn kinase, aggregated, but not unaggregated receptors, and the linker for the activation of T cells (LAT). Phosphatase activity in the rafts is substantially reduced compared to the nonraft region. We use the model to analyze published experiments on the rat basophilic leukemia (RBL)-2H3 cell line that seem to contradict the notion that rafts offer protection. In these experiments IgE was cross-linked with a multivalent antigen and then excess monovalent hapten was added to break-up cross-links. The dephosphorylation of the unaggregated receptor (nonraft associated) and of LAT (raft associated) were then monitored in time and found to decay at similar rates, leading to the conclusion that rafts offer no protection from dephosphorylation. In the model, because the rafts are transient, a protein that is protected while in a raft will be subject to dephosphorylation when the raft breaks up and the protein finds itself in the nonraft region of the membrane. We show that the model is consistent with the receptor and LAT dephosphorylation experiments while still allowing rafts to enhance signaling by providing substantial protection from phosphatases. PMID:23284735

Membrane raft association is a determinant of plasma membrane localization

PubMed Central

Diaz-Rohrer, Blanca B.; Levental, Kandice R.; Simons, Kai; Levental, Ilya

2014-01-01

The lipid raft hypothesis proposes lateral domains driven by preferential interactions between sterols, sphingolipids, and specific proteins as a central mechanism for the regulation of membrane structure and function; however, experimental limitations in defining raft composition and properties have prevented unequivocal demonstration of their functional relevance. Here, we establish a quantitative, functional relationship between raft association and subcellular protein sorting. By systematic mutation of the transmembrane and juxtamembrane domains of a model transmembrane protein, linker for activation of T-cells (LAT), we generated a panel of variants possessing a range of raft affinities. These mutations revealed palmitoylation, transmembrane domain length, and transmembrane sequence to be critical determinants of membrane raft association. Moreover, plasma membrane (PM) localization was strictly dependent on raft partitioning across the entire panel of unrelated mutants, suggesting that raft association is necessary and sufficient for PM sorting of LAT. Abrogation of raft partitioning led to mistargeting to late endosomes/lysosomes because of a failure to recycle from early endosomes. These findings identify structural determinants of raft association and validate lipid-driven domain formation as a mechanism for endosomal protein sorting. PMID:24912166

Recent progress on lipid lateral heterogeneity in plasma membranes: from rafts to submicrometric domains

PubMed Central

Carquin, Mélanie; D'Auria, Ludovic; Pollet, Hélène; Bongarzone, Ernesto R.; Tyteca, Donatienne

2016-01-01

The concept of transient nanometric domains known as lipid rafts has brought interest to reassess the validity of the Singer-Nicholson model of a fluid bilayer for cell membranes. However, this new view is still insufficient to explain the cellular control of surface lipid diversity or membrane deformability. During the past decade, the hypothesis that some lipids form large (submicrometric/mesoscale vs nanometric rafts) and stable (> min vs sec) membrane domains has emerged, largely based on indirect methods. Morphological evidence for stable submicrometric lipid domains, well-accepted for artificial and highly specialized biological membranes, was further reported for a variety of living cells from prokaryotes to yeast and mammalian cells. However, results remained questioned based on limitations of available fluorescent tools, use of poor lipid fixatives, and imaging artifacts due to non-resolved membrane projections. In this review, we will discuss recent evidence generated using powerful and innovative approaches such as lipid-specific toxin fragments that support the existence of submicrometric domains. We will integrate documented mechanisms involved in the formation and maintenance of these domains, and provide a perspective on their relevance on membrane deformability and regulation of membrane protein distribution. PMID:26738447

Pravastatin reverses the membrane cholesterol reorganization induced by myocardial infarction within lipid rafts in CD14(+)/CD16(-) circulating monocytes.

PubMed

Salvary, Thomas; Gambert-Nicot, Ségolène; Brindisi, Marie-Claude; Meneveau, Nicolas; Schiele, François; Séronde, Marie-France; Lorgis, Luc; Zeller, Marianne; Cottin, Yves; Kantelip, Jean-Pierre; Gambert, Philippe; Davani, Siamak

2012-09-01

Large numbers of monocytes are recruited in the infarcted myocardium. Their cell membranes contain cholesterol-rich microdomains called lipids rafts, which participate in numerous signaling cascades. In addition to its cholesterol-lowering effect, pravastatin has several pleiotropic effects and is widely used as secondary prevention treatment after myocardial infarction (MI). The aim of this study was to investigate the effects of pravastatin on the organization of cholesterol within monocyte membrane rafts from patients who had suffered myocardial infarction. Monocytes from healthy donors and acute MI patients were cultured with or without 4μM pravastatin. Lipid rafts were extracted by Lubrol WX, caveolae and flat rafts were separated using a modified sucrose gradient. Cholesterol level and caveolin-1 expression in lipid rafts were determined. In healthy donors, cholesterol was concentrated in flat rafts (63±3 vs 13±1%, p<0.001). While monocytes from MI patients presented similar cholesterol distribution in both caveolae and flat rafts. Cholesterol distribution was higher in flat rafts in healthy donors, compared to MI patients (63±3 vs 41±2%, p<0.001), with less distribution in caveolae (13±1 vs 34±2%, p<0.001). Pravastatin reversed the cholesterol distribution in MI patients cells between flat rafts (41±2 vs 66±3%, p<0.001) and caveolae (34±2 vs 18±1%, p<0.001). In conclusion, MI redistributes cholesterol from flat rafts to caveolae indicating monocyte membrane reorganization. In vitro pravastatin treatment restored basal conditions in MI monocytes, suggesting another effect of statins. Copyright © 2012 Elsevier B.V. All rights reserved.

Critical role of the lipid rafts in caprine herpesvirus type 1 infection in vitro.

PubMed

Pratelli, Annamaria; Colao, Valeriana

2016-01-04

The fusion machinery for herpesvirus entry in the host cells involves the interactions of viral glycoproteins with cellular receptors, although additional viral and cellular domains are required. Extensive areas of the plasma membrane surface consist of lipid rafts organized into cholesterol-rich microdomains involved in signal transduction, protein sorting, membrane transport and in many processes of viruses infection. Because of the extraction of cholesterol leads to disorganization of lipid microdomains and to dissociation of proteins bound to the lipid rafts, we investigated the effect of cholesterol depletion by methyl-β-cyclodextrin (MβCD) on caprine herpesvirus 1 (CpHV.1) in three important phases of virus infection such as binding, entry and post-entry. MβCD treatment did not prejudice virus binding to cells, while a dose-dependent reduction of the virus yield was observed at the virus entry stage, and 30 mM MβCD reduced infectivity evidently. Treatment of MDBK after virus entry revealed a moderate inhibitory effect suggesting that cholesterol is mainly required during virus entry rather than during the post-entry stage. Alteration of the envelope lipid composition affected virus entry and a noticeable reduction in virus infectivity was detected in the presence of 15 mM MβCD. Considering that the recognition of a host cell receptor is a crucial step in the start-up phase of infection, these data are essential for the study of CpHV.1 pathogenesis. To date virus receptors for CpHV.1 have not yet been identified and further investigations are required to state that MβCD treatment affects the expression of the viral receptors. Copyright © 2015 Elsevier B.V. All rights reserved.

Two-Phase Contiguous Supported Lipid Bilayer Model for Membrane Rafts via Polymer Blotting and Stenciling.

PubMed

Richards, Mark J; Daniel, Susan

2017-02-07

The supported lipid bilayer has been portrayed as a useful model of the cell membrane compatible with many biophysical tools and techniques that demonstrate its appeal in learning about the basic features of the plasma membrane. However, some of its potential has yet to be realized, particularly in the area of bilayer patterning and phase/composition heterogeneity. In this work, we generate contiguous bilayer patterns as a model system that captures the general features of membrane domains and lipid rafts. Micropatterned polymer templates of two types are investigated for generating patterned bilayer formation: polymer blotting and polymer lift-off stenciling. While these approaches have been used previously to create bilayer arrays by corralling bilayers patches with various types of boundaries impenetrable to bilayer diffusion, unique to the methods presented here, there are no physical barriers to diffusion. In this work, interfaces between contiguous lipid phases define the pattern shapes, with continuity between them allowing transfer of membrane-bound biomolecules between the phases. We examine effectors of membrane domain stability including temperature and cholesterol content to investigate domain dynamics. Contiguous patterning of supported bilayers as a model of lipid rafts expands the application of the SLB to an area with current appeal and brings with it a useful toolset for characterization and analysis. These combined tools should be helpful to researchers investigating lipid raft dynamics and function and biomolecule partitioning studies. Additionally, this patterning technique may be useful for applications such as bioseparations that exploit differences in lipid phase partitioning or creation of membranes that bind species like viruses preferentially at lipid phase boundaries, to name a few.

Synaptic proteins associate with a sub-set of lipid rafts when isolated from nerve endings at physiological temperature.

PubMed

Gil, Carles; Cubí, Roger; Blasi, Juan; Aguilera, José

2006-10-06

Although the high presence of cholesterol in nerve terminals is well documented, specific roles of this lipid in transmitter release have remained elusive. Since cholesterol is a highly enriched component in the membrane microdomains known as lipid rafts, it is probable that these domains are very important in synaptic function. The extraction of lipid rafts using Brij 98 at 37 degrees C avoids the formation of nonspecific membrane aggregates at low temperature, allowing the isolation of more physiologically relevant lipid rafts. In the present work, we examine, by means of buoyancy analysis in sucrose gradients after solubilization of the membranes with Brij 98 or with Lubrol WX, the presence of proteins involved in exocytosis in detergent-resistant membranes (DRM) using rat brain synaptosomes as a neurological model. Significant proportions of the proteins tested in the present work, which are involved in neurotransmitter release, are found in Brij 98 raft fractions, demonstrating that significant pools of synaptic proteins are segregated in specific parts of the membrane at physiological temperature. On the other hand, Lubrol WX is unable to solubilize the major fraction of the proteins tested. Treatment of synaptosomes with methyl-beta-cyclodextrin (mbetaCD) causes alteration in the buoyancy properties of proteins initially present in Brij- as well as in Lubrol-resistant membranes, indicating the cholesterol-dependency of both kinds of microdomains. Finally, we detect the depolarization-induced enhancement of the cholesterol-dependent association of syntaxin 1 with Brij 98-rafts, under the same conditions in which prolonged neurotransmitter release is stimulated.

The structural role of cholesterol in cell membranes: from condensed bilayers to lipid rafts.

PubMed

Krause, Martin R; Regen, Steven L

2014-12-16

CONSPECTUS: Defining the two-dimensional structure of cell membranes represents one of the most daunting challenges currently facing chemists, biochemists, and biophysicists. In particular, the time-averaged lateral organization of the lipids and proteins that make up these natural enclosures has yet to be established. As the classic Singer-Nicolson model of cell membranes has evolved over the past 40 years, special attention has focused on the structural role played by cholesterol, a key component that represents ca. 30% of the total lipids that are present. Despite extensive studies with model membranes, two fundamental issues have remained a mystery: (i) the mechanism by which cholesterol condenses low-melting lipids by uncoiling their acyl chains and (ii) the thermodynamics of the interaction between cholesterol and high- and low-melting lipids. The latter bears directly on one of the most popular notions in modern cell biology, that is, the lipid raft hypothesis, whereby cholesterol is thought to combine with high-melting lipids to form "lipid rafts" that float in a "sea" of low-melting lipids. In this Account, we first describe a chemical approach that we have developed in our laboratories that has allowed us to quantify the interactions between exchangeable mimics of cholesterol and low- and high-melting lipids in model membranes. In essence, this "nearest-neighbor recognition" (NNR) method involves the synthesis of dimeric forms of these lipids that contain a disulfide moiety as a linker. By means of thiolate-disulfide interchange reactions, equilibrium mixtures of dimers are then formed. These exchange reactions are initiated either by adding dithiothreitol to a liposomal dispersion to generate a small amount of thiol monomer or by including a small amount of thiol monomer in the liposomes at pH 5.0 and then raising the pH to 7.4. We then show how such NNR measurements have allowed us to distinguish between two very different mechanisms that have been

The role of lipid raft translocation of prohibitin in regulation of Akt and Raf-protected apoptosis of HaCaT cells upon ultraviolet B irradiation.

PubMed

Wu, Qiong; Wu, Shiyong

2017-07-01

Prohibitin (PHB) plays a role in regulation of ultraviolet B light (UVB)-induced apoptosis of human keratinocytes, HaCaT cells. The regulatory function of PHB appears to be associated with its lipid raft translocation. However, the detailed mechanism for PHB-mediated apoptosis of these keratinocytes upon UVB irradiation is not clear. In this report, we determined the role of lipid raft translocation of PHB in regulation of UVB-induced apoptosis. Our data show that upon UVB irradiation PHB is translocated from the non-raft membrane to the lipid rafts, which is correlated with a release of both Akt and Raf from membrane. Overexpression of Akt and/or Raf impedes UVB-induced lipid raft translocation of PHB. Immunoprecipitation analysis indicates that UVB alters the interactions among PHB, Akt, and Raf. Reduced expression of PHB leads to a decreased phosphorylation of Akt and ERK, as well as a decreased activity of Akt, and increased apoptosis of the cells upon UVB irradiation. These results suggest that PHB regulates UVB-induced apoptosis of keratinocytes via a mechanism that involves detachment from Akt and Raf on the plasma membrane, and sequential lipid raft translocation. © 2017 Wiley Periodicals, Inc.

Spatial segregation of transport and signalling functions between human endothelial caveolae and lipid raft proteomes

PubMed Central

Sprenger, Richard R.; Fontijn, Ruud D.; van Marle, Jan; Pannekoek, Hans; Horrevoets, Anton J. G.

2006-01-01

Lipid rafts and caveolae are biochemically similar, specialized domains of the PM (plasma membrane) that cluster specific proteins. However, they are morphologically distinct, implying different, possibly complementary functions. Two-dimensional gel electrophoresis preceding identification of proteins by MS was used to compare the relative abundance of proteins in DRMs (detergent-resistant membranes) isolated from HUVEC (human umbilical-vein endothelial cells), and caveolae immunopurified from DRM fractions. Various signalling and transport proteins were identified and additional cell-surface biotinylation revealed the majority to be exposed, demonstrating their presence at the PM. In resting endothelial cells, the scaffold of immunoisolated caveolae consists of only few resident proteins, related to structure [CAV1 (caveolin-1), vimentin] and transport (V-ATPase), as well as the GPI (glycosylphosphatidylinositol)-linked, surface-exposed protein CD59. Further quantitative characterization by immunoblotting and confocal microscopy of well-known [eNOS (endothelial nitric oxide synthase) and CAV1], less known [SNAP-23 (23 kDa synaptosome-associated protein) and BASP1 (brain acid soluble protein 1)] and novel [C8ORF2 (chromosome 8 open reading frame 2)] proteins showed different subcellular distributions with none of these proteins being exclusive to either caveolae or DRM. However, the DRM-associated fraction of the novel protein C8ORF2 (∼5% of total protein) associated with immunoseparated caveolae, in contrast with the raft protein SNAP-23. The segregation of caveolae from lipid rafts was visually confirmed in proliferating cells, where CAV1 was spatially separated from eNOS, SNAP-23 and BASP1. These results provide direct evidence for the previously suggested segregation of transport and signalling functions between specialized domains of the endothelial plasma membrane. PMID:16886909

Second-Hand Cigarette Smoke Impairs Bacterial Phagocytosis in Macrophages by Modulating CFTR Dependent Lipid-Rafts

PubMed Central

Ni, Inzer; Ji, Changhoon; Vij, Neeraj

2015-01-01

Introduction First/Second-hand cigarette-smoke (FHS/SHS) exposure weakens immune defenses inducing chronic obstructive pulmonary disease (COPD) but the underlying mechanisms are not fully understood. Hence, we evaluated if SHS induced changes in membrane/lipid-raft (m-/r)-CFTR (cystic fibrosis transmembrane conductance regulator) expression/activity is a potential mechanism for impaired bacterial phagocytosis in COPD. Methods RAW264.7 murine macrophages were exposed to freshly prepared CS-extract (CSE) containing culture media and/or Pseudomonas-aeruginosa-PA01-GFP for phagocytosis (fluorescence-microscopy), bacterial survival (colony-forming-units-CFU), and immunoblotting assays. The CFTR-expression/activity and lipid-rafts were modulated by transient-transfection or inhibitors/inducers. Next, mice were exposed to acute/sub-chronic-SHS or room-air (5-days/3-weeks) and infected with PA01-GFP, followed by quantification of bacterial survival by CFU-assay. Results We investigated the effect of CSE treatment on RAW264.7 cells infected by PA01-GFP and observed that CSE treatment significantly (p<0.01) inhibits PA01-GFP phagocytosis as compared to the controls. We also verified this in murine model, exposed to acute/sub-chronic-SHS and found significant (p<0.05, p<0.02) increase in bacterial survival in the SHS-exposed lungs as compared to the room-air controls. Next, we examined the effect of impaired CFTR ion-channel-activity on PA01-GFP infection of RAW264.7 cells using CFTR172-inhibitor and found no significant change in phagocytosis. We also similarly evaluated the effect of a CFTR corrector-potentiator compound, VRT-532, and observed no significant rescue of CSE impaired PA01-GFP phagocytosis although it significantly (p<0.05) decreases CSE induced bacterial survival. Moreover, induction of CFTR expression in macrophages significantly (p<0.03) improves CSE impaired PA01-GFP phagocytosis as compared to the control. Next, we verified the link between m

Use of quantitative optical imaging to examine the role of cholesterol-rich lipid raft microdomains in the migration of breast cancer cells

NASA Astrophysics Data System (ADS)

You, Minghai; Chen, Jianling; Wang, Shaobing; Dong, Shiqing; Wang, Yuhua; Xie, Shusen; Wang, Zhengchao; Yang, Hongqin

2018-04-01

Lipid rafts have been extensively studied and shown to be involved in many cancers, including breast cancer. However, the exact role of lipid rafts in the migration of breast cancer cells remains unclear. This study was designed to examine lipid rafts (cholesterol) in the plasma membrane of breast cancer cells (MDA-MB-231 and MCF-7) and normal breast epithelial cells (MCF-10A) through generalized polarization values, and further investigate the role of cholesterol-rich lipid rafts in the migration of breast cancer cells. The results showed that the plasma membrane in breast cancer cells was more orderly than that in normal epithelial cells; this was correlated with expression changes of matrix metallopeptidase 9 (MMP-9) and urokinase-type plasminogen activator receptor (uPAR), the markers of cancer cell migration. Moreover, the breast cancer cells were more sensitive to the reagent that induced cholesterol depletion than the normal breast epithelial cells, while the capacity of cancer cells to migrate decreased significantly according to changes in MMP-9 and uPAR expression. To our best knowledge, this is the first demonstration of the relationship between cholesterol-rich lipid rafts and the migration of breast cancer cells; it could be useful for the prevention of breast cancer and early treatment through reduction of the level of cholesterol in the plasma membrane of the cells.

Reconstituted high-density lipoprotein suppresses leukocyte NADPH oxidase activation by disrupting lipid rafts.

PubMed

Peshavariya, Hitesh; Dusting, Gregory J; Di Bartolo, Belinda; Rye, Kerry-Anne; Barter, Philip J; Jiang, Fan

2009-08-01

Reconstituted discoidal high-density lipoprotein (rHDL) has potent vascular protective actions. Native HDL suppresses cellular generation of reactive oxygen species, whereas this antioxidant effect of rHDL is less clear. This study examined the effects of rHDL on NADPH oxidase, a major source of cellular superoxide generation, in both leukocytes and human umbilical vein endothelial cells. Superoxide was measured with lucigenin-enhanced chemiluminescence. Expression of NADPH oxidase sub-units was determined by real-time PCR. Pre-treatment of HL-60 cells with rHDL (10 and 25 microM) for 1 h significantly reduced phorbol 12-myristate 13-acetate-stimulated superoxide production. Treatment with rHDL for up to 24 h did not change the mRNA expression of NADPH oxidase sub-units. In HL-60 cells, depletion of cholesterol from the plasma membrane by methyl-beta-cyclodextrin mimicked the effect of rHDL, whereas cholesterol repletion blunted the effects of rHDL. Treatment with rHDL induced disruption of the lipid raft structures and blunted PMA-induced redistribution of p47phox into lipid rafts. In contrast, treatment of endothelial cells with rHDL for up to 18 h had no effect on either basal or tumour necrosis factor-alpha-stimulated NADPH oxidase activity, but markedly suppressed the cytokine-induced expression of proinflammatory adhesion molecules. The results suggest that rHDL inhibits NADPH oxidase activation in leukocytes, probably by interrupting the assembly of NADPH oxidase sub-units at the lipid rafts. This effect may contribute to the vascular protective actions of rHDL against inflammation-mediated oxidative damage.

Recruitment of α7 nicotinic acetylcholine receptor to caveolin-1-enriched lipid rafts is required for nicotine-enhanced Escherichia coli K1 entry into brain endothelial cells.

PubMed

Chi, Feng; Wang, Lin; Zheng, Xueye; Jong, Ambrose; Huang, Sheng-He

2011-08-01

We investigate how the α7 nicotinic acetylcholine receptor (α7 nAChR), an essential regulator of inflammation, contributes to the α7 agonist nicotine-enhanced Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMECs) through lipid rafts/caveolae-mediated signaling. α7 nAChR-mediated signaling and bacterial invasion were defined by lipid raft fractionation, immunofluorescence microscopy and siRNA knockdown. Nicotine-enhanced bacterial invasion was dose-dependently inhibited by two raft-disrupting agents, nystatin and filipin. Significant accumulation of the lipid raft marker GM3 was observed in HBMEC induced by E. coli K1 and nicotine. The recruitment of α7 nAChR and related signaling molecules, including vimentin, and Erk1/2, to caveolin-1 enriched lipid rafts was increased upon treatment with E44 or E44 plus nicotine. Erk1/2 activation (phosphorylation), which is required for α7 nAChR-mediated signaling and E44 invasion, was associated with lipid rafts and nicotine-enhanced bacterial infection. Furthermore, E44 invasion, E44/nicotine-induced activation of Erk1/2 and clustering of α7 nAChR and caveolin-1 was specifically blocked by both siRNAs. α7 nAChR-mediated signaling through lipid rafts/caveolae is required for nicotine-enhanced E. coli K1 invasion of HBMEC.

Roles of Raft-Anchored Adaptor Cbp/PAG1 in Spatial Regulation of c-Src Kinase

PubMed Central

Oneyama, Chitose; Suzuki, Takashi; Okada, Masato

2014-01-01

The tyrosine kinase c-Src is upregulated in numerous human cancers, implying a role for c-Src in cancer progression. Previously, we have shown that sequestration of activated c-Src into lipid rafts via a transmembrane adaptor, Cbp/PAG1, efficiently suppresses c-Src-induced cell transformation in Csk-deficient cells, suggesting that the transforming activity of c-Src is spatially regulated via Cbp in lipid rafts. To dissect the molecular mechanisms of the Cbp-mediated regulation of c-Src, a combined analysis was performed that included mathematical modeling and in vitro experiments in a c-Src- or Cbp-inducible system. c-Src activity was first determined as a function of c-Src or Cbp levels, using focal adhesion kinase (FAK) as a crucial c-Src substrate. Based on these experimental data, two mathematical models were constructed, the sequestration model and the ternary model. The computational analysis showed that both models supported our proposal that raft localization of Cbp is crucial for the suppression of c-Src function, but the ternary model, which includes a ternary complex consisting of Cbp, c-Src, and FAK, also predicted that c-Src function is dependent on the lipid-raft volume. Experimental analysis revealed that c-Src activity is elevated when lipid rafts are disrupted and the ternary complex forms in non-raft membranes, indicating that the ternary model accurately represents the system. Moreover, the ternary model predicted that, if Cbp enhances the interaction between c-Src and FAK, Cbp could promote c-Src function when lipid rafts are disrupted. These findings underscore the crucial role of lipid rafts in the Cbp-mediated negative regulation of c-Src-transforming activity, and explain the positive role of Cbp in c-Src regulation under particular conditions where lipid rafts are perturbed. PMID:24675741

Revealing the Raft Domain Organization in the Plasma Membrane by Single-Molecule Imaging of Fluorescent Ganglioside Analogs.

PubMed

Suzuki, Kenichi G N; Ando, Hiromune; Komura, Naoko; Konishi, Miku; Imamura, Akihiro; Ishida, Hideharu; Kiso, Makoto; Fujiwara, Takahiro K; Kusumi, Akihiro

2018-01-01

Gangliosides have been implicated in a variety of physiological processes, particularly in the formation and function of raft domains in the plasma membrane. However, the scarcity of suitable fluorescent ganglioside analogs had long prevented us from determining exactly how gangliosides perform their functions in the live-cell plasma membrane. With the development of new fluorescent ganglioside analogs, as described by Komura et al. (2017), this barrier has been broken. We can now address the dynamic behaviors of gangliosides in the live-cell plasma membrane, using fluorescence microscopy, particularly by single-fluorescent molecule imaging and tracking. Single-molecule tracking of fluorescent GM1 and GM3 revealed that these molecules are transiently and dynamically recruited to monomers (monomer-associated rafts) and homodimer rafts of the raftophilic GPI-anchored protein CD59 in quiescent cells, with exponential residency times of 12 and 40ms, respectively, in a manner dependent on raft-lipid interactions. Upon CD59 stimulation, which induces CD59-cluster signaling rafts, the fluorescent GM1 and GM3 analogs were recruited to the signaling rafts, with a lifetime of 48ms. These results represent the first direct evidence that GPI-anchored receptors and gangliosides interact in a cholesterol-dependent manner. Furthermore, they show that gangliosides continually move in and out of rafts that contain CD59 in an extremely dynamic manner, with much higher frequency than expected previously. Such studies would not have been possible without fluorescent ganglioside probes, which exhibit native-like behavior and single-molecule tracking. In this chapter, we review the methods for single-molecule tracking of fluorescent ganglioside analogs and the results obtained by applying these methods. © 2018 Elsevier Inc. All rights reserved.

Suppressing the formation of lipid raft-associated Rac1/PI3K/Akt signaling complexes by curcumin inhibits SDF-1α-induced invasion of human esophageal carcinoma cells.

PubMed

Lin, Meng-Liang; Lu, Yao-Cheng; Chen, Hung-Yi; Lee, Chuan-Chun; Chung, Jing-Gung; Chen, Shih-Shun

2014-05-01

Stromal cell-derived factor-1α (SDF-1α) is a ligand for C-X-C chemokine receptor type 4 (CXCR4), which contributes to the metastasis of cancer cells by promoting cell migration. Here, we show that the SDF-1α/CXCR4 axis can significantly increase invasion of esophageal carcinoma (EC) cells. We accomplished this by examining the effects of CXCR4 knockdown as well as treatment with a CXCR4-neutralizing antibody and the CXCR4-specific inhibitor AMD3100. Curcumin suppressed SDF-1α-induced cell invasion and matrix metalloproteinase-2 (MMP-2) promoter activity, cell surface localization of CXCR4 at lipid rafts, and lipid raft-associated ras-related C3 botulinum toxin substrate 1 (Rac1)/phosphatidylinositol 3-kinase (PI3K) p85α/Akt signaling. Curcumin inhibited SDF-1α-induced cell invasion by suppressing the Rac1-PI3K signaling complex at lipid rafts but did not abrogate lipid raft formation. We further demonstrate that the attenuation of lipid raft-associated Rac1 activity by curcumin was critical for the inhibition of SDF-1α-induced PI3K/Akt/NF-κB activation, cell surface localization of CXCR4 at lipid rafts, MMP-2 promoter activity, and cell invasion. Collectively, our results indicate that curcumin inhibits SDF-1α-induced EC cell invasion by suppressing the formation of the lipid raft-associated Rac1-PI3K-Akt signaling complex, the localization of CXCR4 with lipid rafts at the cell surface, and MMP-2 promoter activity, likely through the inhibition of Rac1 activity. © 2012 Wiley Periodicals, Inc.

Docosahexaenoic acid modifies the clustering and size of lipid rafts and the lateral organization and surface expression of MHC class I of EL4 cells.

PubMed

Shaikh, Saame Raza; Rockett, Benjamin Drew; Salameh, Muhammad; Carraway, Kristen

2009-09-01

An emerging molecular mechanism by which docosahexaenoic acid (DHA) exerts its effects is modification of lipid raft organization. The biophysical model, based on studies with liposomes, shows that DHA avoids lipid rafts because of steric incompatibility between DHA and cholesterol. The model predicts that DHA does not directly modify rafts; rather, it incorporates into nonrafts to modify the lateral organization and/or conformation of membrane proteins, such as the major histocompatibility complex (MHC) class I. Here, we tested predictions of the model at a cellular level by incorporating oleic acid, eicosapentaenoic acid (EPA), and DHA, compared with a bovine serum albumin (BSA) control, into the membranes of EL4 cells. Quantitative microscopy showed that DHA, but not EPA, treatment, relative to the BSA control diminished lipid raft clustering and increased their size. Approximately 30% of DHA was incorporated directly into rafts without changing the distribution of cholesterol between rafts and nonrafts. Quantification of fluorescence colocalization images showed that DHA selectively altered MHC class I lateral organization by increasing the fraction of the nonraft protein into rafts compared with BSA. Both DHA and EPA treatments increased antibody binding to MHC class I compared with BSA. Antibody titration showed that DHA and EPA did not change MHC I conformation but increased total surface levels relative to BSA. Taken together, our findings are not in agreement with the biophysical model. Therefore, we propose a model that reconciles contradictory viewpoints from biophysical and cellular studies to explain how DHA modifies lipid rafts on several length scales. Our study supports the notion that rafts are an important target of DHA's mode of action.

Proteomic Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling.

PubMed

Chowdhury, Saiful M; Zhu, Xuewei; Aloor, Jim J; Azzam, Kathleen M; Gabor, Kristin A; Ge, William; Addo, Kezia A; Tomer, Kenneth B; Parks, John S; Fessler, Michael B

2015-07-01

Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1(-/-) macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1(+/+) and Abca1(-/-) macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1(+/+) macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1(-/-) rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response. © 2015 by The American Society for Biochemistry and

Proteomic Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling*

PubMed Central

Chowdhury, Saiful M.; Zhu, Xuewei; Aloor, Jim J.; Azzam, Kathleen M.; Gabor, Kristin A.; Ge, William; Addo, Kezia A.; Tomer, Kenneth B.; Parks, John S.; Fessler, Michael B.

2015-01-01

Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1−/− macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1+/+ and Abca1−/− macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1+/+ macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1−/− rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response. PMID:25910759

Light on fluorescent lipids in rafts: a lesson from model membranes.

PubMed

Kahya, Nicoletta

2010-09-15

Tracking fluorescent lipids in cellular membranes has been applied for decades to shed light on membrane trafficking, sorting, endocytosis and exocytosis, viral entry, and to understand the functional relevance of membrane heterogeneity, phase separation and lipid rafts. However, fluorescent probes may display different organizing behaviour from their corresponding endogenous lipids. A full characterization of these probes is therefore required for proper interpretation of fluorescence microscopy data in complex membrane systems. Model membrane studies provide essential clues that guide us to design and interpret our experiments, help us to avoid pitfalls and resolve artefacts in complex cellular environments. In the present issue of the Biochemical Journal, Juhasz, Davis and Sharom demonstrate the importance of testing lipid probes systematically in heterogeneous model membranes of specific composition and well-defined thermodynamic properties. The phase-partitioning behaviour of fluorescent probes, alone and/or in combination, cannot simply be assumed, but has to be fully characterized.

Influenza A Virus Hemagglutinin is Required for the Assembly of Viral Components Including Bundled vRNPs at the Lipid Raft.

PubMed

Takizawa, Naoki; Momose, Fumitaka; Morikawa, Yuko; Nomoto, Akio

2016-09-10

The influenza glycoproteins, hemagglutinin (HA) and neuraminidase (NA), which are associated with the lipid raft, have the potential to initiate virion budding. However, the role of these viral proteins in infectious virion assembly is still unclear. In addition, it is not known how the viral ribonucleoprotein complex (vRNP) is tethered to the budding site. Here, we show that HA is necessary for the efficient progeny virion production and vRNP packaging in the virion. We also found that the level of HA does not affect the bundling of the eight vRNP segments, despite reduced virion production. Detergent solubilization and a subsequent membrane flotation analysis indicated that the accumulation of nucleoprotein, viral polymerases, NA, and matrix protein 1 (M1) in the lipid raft fraction was delayed without HA. Based on our results, we inferred that HA plays a role in the accumulation of viral components, including bundled vRNPs, at the lipid raft.

Na/K-ATPase regulates bovine sperm capacitation through raft- and non-raft-mediated signaling mechanisms.

PubMed

Rajamanickam, Gayathri D; Kastelic, John P; Thundathil, Jacob C

2017-11-01

Highly dynamic lipid microdomains (rafts) in the sperm plasma membrane contain several signaling proteins that regulate sperm capacitation. Na/K-ATPase isoforms (testis-specific isoform ATP1A4 and ubiquitous isoform ATP1A1) are abundant in bovine sperm plasma membrane. We previously reported that incubation of bovine sperm with ouabain, a specific Na/K-ATPase ligand, induced tyrosine phosphorylation of several sperm proteins during capacitation. The objective of this study was to investigate the roles of lipid rafts and non-rafts in Na/K-ATPase enzyme activity and signaling during bovine sperm capacitation. Content of ATP1A4 and, to a lesser extent, ATP1A1 was increased in raft and non-raft fractions of capacitated sperm, although non-raft enzyme activities of both isoforms were higher than the corresponding activities in rafts from capacitated sperm. Yet, ATP1A4 was the predominant isoform responsible for total Na/K-ATPase activity in both rafts and non-rafts. A comparative increase in phosphorylation of signaling molecules was observed in both raft (CAV1) and non-raft (EGFR and ERK1/2) membrane fractions during capacitation. Although SRC was phosphorylated in both membrane fractions, the non-raft fraction possessed more of this activated form. We also inferred, by immunoprecipitation, that ATP1A4 interacted with CAV1 and EGFR in the raft fraction, whereas interactions of ATP1A4 with SRC, EGFR, and ERK1/2 occurred in the non-raft fraction of ouabain-capacitated sperm; conversely, ATP1A1 interacted only with CAV1 in both fractions of uncapacitated and capacitated sperm. In conclusion, both raft and non-raft cohorts of Na/K-ATPase isoforms contributed to phosphorylation of signaling molecules during bovine sperm capacitation. © 2017 Wiley Periodicals, Inc.

Oxidized LDL lipids increase β-amyloid production by SH-SY5Y cells through glutathione depletion and lipid raft formation

PubMed Central

Dias, Irundika H.K.; Mistry, Jayna; Fell, Shaun; Reis, Ana; Spickett, Corinne M.; Polidori, Maria C.; Lip, Gregory Y.H.; Griffiths, Helen R.

2014-01-01

Elevated total cholesterol in midlife has been associated with increased risk of dementia in later life. We have previously shown that low-density lipoprotein (LDL) is more oxidized in the plasma of dementia patients, although total cholesterol levels are not different from those of age-matched controls. β-Amyloid (Aβ) peptide, which accumulates in Alzheimer disease (AD), arises from the initial cleavage of amyloid precursor protein by β-secretase-1 (BACE1). BACE1 activity is regulated by membrane lipids and raft formation. Given the evidence for altered lipid metabolism in AD, we have investigated a mechanism for enhanced Aβ production by SH-SY5Y neuronal-like cells exposed to oxidized LDL (oxLDL). The viability of SH-SY5Y cells exposed to 4 μg oxLDL and 25 µM 27-hydroxycholesterol (27OH-C) was decreased significantly. Lipids, but not proteins, extracted from oxLDL were more cytotoxic than oxLDL. In parallel, the ratio of reduced glutathione (GSH) to oxidized glutathione was decreased at sublethal concentrations of lipids extracted from native and oxLDL. GSH loss was associated with an increase in acid sphingomyelinase (ASMase) activity and lipid raft formation, which could be inhibited by the ASMase inhibitor desipramine. 27OH-C and total lipids from LDL and oxLDL independently increased Aβ production by SH-SY5Y cells, and Aβ accumulation could be inhibited by desipramine and by N-acetylcysteine. These data suggest a mechanism whereby oxLDL lipids and 27OH-C can drive Aβ production by GSH depletion, ASMase-driven membrane remodeling, and BACE1 activation in neuronal cells. PMID:25048970

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

PubMed

Kardash, Maria E; Dzuba, Sergei A

2017-05-25

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.

High-density lipoprotein and apolipoprotein A-I inhibit palmitate-induced translocation of toll-like receptor 4 into lipid rafts and inflammatory cytokines in 3T3-L1 adipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamada, Hodaka; Umemoto, Tomio; Kawano, Mikihiko

Saturated fatty acids (SFAs) activate toll-like receptor 4 (TLR4) signal transduction in macrophages and are involved in the chronic inflammation accompanying obesity. High-density lipoprotein (HDL) and apolipoprotein A-I (apoA-I) produce anti-inflammatory effects via reverse cholesterol transport. However, the underlying mechanisms by which HDL and apoA-I inhibit inflammatory responses in adipocytes remain to be determined. Here we examined whether palmitate increases the translocation of TLR4 into lipid rafts and whether HDL and apoA-I inhibit inflammation in adipocytes. Palmitate exposure (250 μM, 24 h) increased interleukin-6 and tumor necrosis factor-α gene expressions and translocation of TLR4 into lipid rafts in 3T3-L1 adipocytes. Pretreatment withmore » HDL and apoA-I (50 μg/mL, 6 h) suppressed palmitate-induced inflammatory cytokine expression and TLR4 translocation into lipid rafts. Moreover, HDL and apoA-I inhibited palmitate-induced phosphorylation of nuclear factor-kappa B. HDL showed an anti-inflammatory effect via ATP-binding cassette transporter G1 and scavenger receptor class B, member 1, whereas apoA-I showed an effect via ATP-binding cassette transporter A1. These results demonstrated that HDL and apoA-I reduced palmitate-potentiated TLR4 trafficking into lipid rafts and its related inflammation in adipocytes via these specific transporters. - Highlights: • Palmitate induces TLR4 translocation into lipid rafts in 3T3-L1 adipocytes. • Raft disruption by MβCD inhibits lipid raft formation. • HDL and apoA-I inhibit palmitate-induced translocation of TLR4 into lipid rafts. • Anti-inflammatory effects of HDL and apoA-I occur via specific transporters.« less

Endothelial microparticle formation by angiotensin II is mediated via Ang II receptor type I/NADPH oxidase/ Rho kinase pathways targeted to lipid rafts.

PubMed

Burger, Dylan; Montezano, Augusto C; Nishigaki, Nobuhiro; He, Ying; Carter, Anthony; Touyz, Rhian M

2011-08-01

Circulating microparticles are increased in cardiovascular disease and may themselves promote oxidative stress and inflammation. Molecular mechanisms underlying their formation and signaling are unclear. We investigated the role of reactive oxygen species (ROS), Rho kinase, and lipid rafts in microparticle formation and examined their functional significance in endothelial cells (ECs). Microparticle formation from angiotensin II (Ang II)-stimulated ECs and apolipoprotein E(-/-) mice was assessed by annexin V or by CD144 staining and electron microscopy. Ang II promoted microparticle formation and increased EC O(2)(-) generation and Rho kinase activity. Ang II-stimulated effects were inhibited by irbesartan (Ang II receptor type I blocker) and fasudil (Rho kinase inhibitor). Methyl-β-cyclodextrin and nystatin, which disrupt lipid rafts/caveolae, blocked microparticle release. Functional responses, assessed in microparticle-stimulated ECs, revealed increased O(2)(-) production, enhanced vascular cell adhesion molecule/platelet-EC adhesion molecule expression, and augmented macrophage adhesion. Inhibition of epidermal growth factor receptor blocked the prooxidative and proinflammatory effects of microparticles. In vitro observations were confirmed in apolipoprotein E(-/-) mice, which displayed vascular inflammation and high levels of circulating endothelial microparticles, effects that were reduced by apocynin. We demonstrated direct actions of Ang II on endothelial microparticle release, mediated through NADPH oxidase, ROS, and Rho kinase targeted to lipid rafts. Microparticles themselves stimulated endothelial ROS formation and inflammatory responses. Our findings suggest a feedforward system whereby Ang II promotes EC injury through its own endothelial-derived microparticles.

Absence of death receptor translocation into lipid rafts in acquired TRAIL-resistant NSCLC cells.

PubMed

Ouyang, Wen; Yang, Chunxu; Zhang, Simin; Liu, Yu; Yang, Bo; Zhang, Junhong; Zhou, Fuxiang; Zhou, Yunfeng; Xie, Conghua

2013-02-01

Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a major limitation for its clinical use. The mechanisms of TRAIL resistance have been mostly studied in the context of cell lines that are intrinsically resistant to TRAIL. However, little is known about the molecular alterations that contribute to the development of acquired resistance during treatment with TRAIL. In this study, we established H460R, an isogenic cell line with acquired TRAIL resistance, from the TRAIL‑sensitive human lung cancer cell line H460 to investigate the mechanisms of acquired resistance. The acquired TRAIL‑resistant H460R cells remained sensitive to cisplatin. The mRNA and protein expression levels of death receptor 4 (DR4) and death receptor 5 (DR5) were not altered in either of the TRAIL-treated cell lines. Nevertheless, tests in which the DR4 or DR5 gene was overexpressed or silenced suggest that death receptor expression is necessary but not sufficient for TRAIL‑induced apoptosis. Compared with parental TRAIL-sensitive H460 cells, H460R cells showed a decreased TRAIL-induced translocation of DR4/DR5 into lipid rafts. Further studies showed that nystatin partially prevented lipid raft aggregation and DR4 and DR5 clustering and reduced apoptosis in H460 cells again. Analysis of apoptotic molecules showed that more pro-caspase-8, FADD, caspase-3 and Bid, but less cFLIP in H460 cells than in H460R cells. Our findings suggest that the lack of death receptor redistribution negatively impacts DISC assembly in lipid rafts, which at least partially leads to the development of acquired resistance to TRAIL in H460R cells.

Prion Protein-mediated Toxicity of Amyloid-β Oligomers Requires Lipid Rafts and the Transmembrane LRP1*

PubMed Central

Rushworth, Jo V.; Griffiths, Heledd H.; Watt, Nicole T.; Hooper, Nigel M.

2013-01-01

Soluble oligomers of the amyloid-β (Aβ) peptide cause neurotoxicity, synaptic dysfunction, and memory impairments that underlie Alzheimer disease (AD). The cellular prion protein (PrPC) was recently identified as a high affinity neuronal receptor for Aβ oligomers. We report that fibrillar Aβ oligomers recognized by the OC antibody, which have been shown to correlate with the onset and severity of AD, bind preferentially to cells and neurons expressing PrPC. The binding of Aβ oligomers to cell surface PrPC, as well as their downstream activation of Fyn kinase, was dependent on the integrity of cholesterol-rich lipid rafts. In SH-SY5Y cells, fluorescence microscopy and co-localization with subcellular markers revealed that the Aβ oligomers co-internalized with PrPC, accumulated in endosomes, and subsequently trafficked to lysosomes. The cell surface binding, internalization, and downstream toxicity of Aβ oligomers was dependent on the transmembrane low density lipoprotein receptor-related protein-1 (LRP1). The binding of Aβ oligomers to cell surface PrPC impaired its ability to inhibit the activity of the β-secretase BACE1, which cleaves the amyloid precursor protein to produce Aβ. The green tea polyphenol (−)-epigallocatechin gallate and the red wine extract resveratrol both remodeled the fibrillar conformation of Aβ oligomers. The resulting nonfibrillar oligomers displayed significantly reduced binding to PrPC-expressing cells and were no longer cytotoxic. These data indicate that soluble, fibrillar Aβ oligomers bind to PrPC in a conformation-dependent manner and require the integrity of lipid rafts and the transmembrane LRP1 for their cytotoxicity, thus revealing potential targets to alleviate the neurotoxic properties of Aβ oligomers in AD. PMID:23386614

Differential association of the Na+/H+ Exchanger Regulatory Factor (NHERF) family of adaptor proteins with the raft- and the non-raft brush border membrane fractions of NHE3.

PubMed

Sultan, Ayesha; Luo, Min; Yu, Qin; Riederer, Brigitte; Xia, Weiliang; Chen, Mingmin; Lissner, Simone; Gessner, Johannes E; Donowitz, Mark; Yun, C Chris; deJonge, Hugo; Lamprecht, Georg; Seidler, Ursula

2013-01-01

Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50), NHERF2 (E3KARP), and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM. Detergent resistant membranes ("lipid rafts") were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO2/HCO3(-) mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs. © 2013 S. Karger AG, Basel.

Oxidative Stress in HIV Infection and Alcohol Use: Role of Redox Signals in Modulation of Lipid Rafts and ATP-Binding Cassette Transporters.

PubMed

Thangavel, Samikkannu; Mulet, Carmen T; Atluri, Venkata S R; Agudelo, Marisela; Rosenberg, Rhonda; Devieux, Jessy G; Nair, Madhavan P N

2018-02-01

Human immunodeficiency virus (HIV) infection induces oxidative stress and alcohol use accelerates disease progression, subsequently causing immune dysfunction. However, HIV and alcohol impact on lipid rafts-mediated immune dysfunction remains unknown. In this study, we investigate the modulation by which oxidative stress induces reactive oxygen species (ROS) affecting redox expression, lipid rafts caveiloin-1, ATP-binding cassette (ABC) transporters, and transcriptional sterol regulatory element-binding protein (SREBP) gene and protein modification and how these mechanisms are associated with arachidonic acid (AA) metabolites in HIV positive alcohol users, and how they escalate immune dysfunction. In both alcohol using HIV-positive human subjects and in vitro studies of alcohol with HIV-1 gp120 protein in peripheral blood mononuclear cells, increased ROS production significantly affected redox expression in glutathione synthetase (GSS), super oxide dismutase (SOD), and glutathione peroxidase (GPx), and subsequently impacted lipid rafts Cav-1, ABC transporters ABCA1, ABCG1, ABCB1, and ABCG4, and SREBP transcription. The increased level of rate-limiting enzyme 3-hydroxy-3-methylglutaryl HMG-CoA reductase (HMGCR), subsequently, inhibited 7-dehydrocholesterol reductase (DHCR-7). Moreover, the expression of cyclooxygenase-2 (COX-2) and lipoxygenase-5 (5-LOX) mRNA and protein modification tentatively increased the levels of prostaglandin E2 synthases (PGE 2 ) in plasma when compared with either HIV or alcohol alone. This article suggests for the first time that the redox inhibition affects lipid rafts, ABC-transporter, and SREBP transcription and modulates AA metabolites, serving as an important intermediate signaling network during immune cell dysfunction in HIV-positive alcohol users. These findings indicate that HIV infection induces oxidative stress and redox inhibition, affecting lipid rafts and ABC transports, subsequently upregulating AA metabolites and leading to

The structure of the CD3 ζζ transmembrane dimer in POPC and raft-like lipid bilayer: a molecular dynamics study.

PubMed

Petruk, Ariel Alcides; Varriale, Sonia; Coscia, Maria Rosaria; Mazzarella, Lelio; Merlino, Antonello; Oreste, Umberto

2013-11-01

Plasma membrane lipids significantly affect assembly and activity of many signaling networks. The present work is aimed at analyzing, by molecular dynamics simulations, the structure and dynamics of the CD3 ζζ dimer in palmitoyl-oleoyl-phosphatidylcholine bilayer (POPC) and in POPC/cholesterol/sphingomyelin bilayer, which resembles the raft membrane microdomain supposed to be the site of the signal transducing machinery. Both POPC and raft-like environment produce significant alterations in structure and flexibility of the CD3 ζζ with respect to nuclear magnetic resonance (NMR) model: the dimer is more compact, its secondary structure is slightly less ordered, the arrangement of the Asp6 pair, which is important for binding to the Arg residue in the alpha chain of the T cell receptor (TCR), is stabilized by water molecules. Different interactions of charged residues with lipids at the lipid-cytoplasm boundary occur when the two environments are compared. Furthermore, in contrast to what is observed in POPC, in the raft-like environment correlated motions between transmembrane and cytoplasmic regions are observed. Altogether the data suggest that when the TCR complex resides in the raft domains, the CD3 ζζ dimer assumes a specific conformation probably necessary to the correct signal transduction. © 2013.

Identification of Novel Raft Marker Protein, FlotP in Bacillus anthracis.

PubMed

Somani, Vikas K; Aggarwal, Somya; Singh, Damini; Prasad, Tulika; Bhatnagar, Rakesh

2016-01-01

Lipid rafts are dynamic, nanoscale assemblies of specific proteins and lipids, distributed heterogeneously on eukaryotic membrane. Flotillin-1, a conserved eukaryotic raft marker protein (RMP) harbor SPFH (Stomatin, Prohibitin, Flotillin, and HflK/C) and oligomerization domains to regulate various cellular processes through its interactions with other signaling or transport proteins. Rafts were thought to be absent in prokaryotes hitherto, but recent report of its presence and significance in physiology of Bacillus subtilis prompted us to investigate the same in pathogenic bacteria (PB) also. In prokaryotes, proteins of SPFH2a subfamily show highest identity to SPFH domain of Flotillin-1. Moreover, bacterial genome organization revealed that Flotillin homolog harboring SPFH2a domain exists in an operon with an upstream gene containing NFeD domain. Here, presence of RMP in PB was initially investigated in silico by analyzing the presence of SPFH2a, oligomerization domains in the concerned gene and NfeD domain in the adjacent upstream gene. After investigating 300 PB, four were found to harbor RMP. Among them, domains of Bas0525 (FlotP) of Bacillus anthracis (BA) showed highest identity with characteristic domains of RMP. Considering the global threat of BA as the bioterror agent, it was selected as a model for further in vitro characterization of rafts in PB. In silico and in vitro analysis showed significant similarity of FlotP with numerous attributes of Flotillin-1. Its punctate distribution on membrane with exclusive localization in detergent resistant membrane fraction; strongly favors presence of raft with RMP FlotP in BA. Furthermore, significant effect of Zaragozic acid (ZA), a raft associated lipid biosynthesis inhibitor, on several patho-physiological attributes of BA such as growth, morphology, membrane rigidity etc., were also observed. Specifically, a considerable decrease in membrane rigidity, strongly recommended presence of an unknown raft associated

Distribution of C16:0, C18:0, C24:1, and C24:0 sulfatides in central nervous system lipid rafts by quantitative ultra-high-pressure liquid chromatography tandem mass spectrometry.

PubMed

Moyano, Ana Lis; Li, Guannan; Lopez-Rosas, Aurora; Månsson, Jan-Eric; van Breemen, Richard B; Givogri, Maria Irene

2014-12-15

Sulfated galactosylceramides (sulfatides) are glycosphingolipids associated with cholesterol- and sphingolipid-enriched membrane microdomains (lipid rafts) and are highly expressed in brain tissue. Although it is known that sulfatide species show heterogeneity in their fatty acid acyl group composition throughout brain development, their lipid raft distribution and biological relevance is poorly understood. We validated a fast and sensitive ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method to measure developmentally regulated sulfatide species (C16:0, C18:0, C24:1, and C24:0) in central nervous system (CNS) lipid rafts isolated without using detergent. Our UHPLC-MS/MS assay showed good accuracy and precision with a linear range of 5 to 1,000 nM for C18:0 and C24:1 sulfatides and 10 to 1,000 nM for C16:0 and C24:0 sulfatides. We applied this quantitative analysis to detergent-free lipid rafts isolated from wild-type mice and arylsulfatase A-deficient (ASA knockout) mice that accumulate sulfatides. All four sulfatide species were more abundant in raft membranes than in non-raft membranes, with a significant increase in lipid rafts isolated from ASA knockout mice. This is the first description of an analytical method to study these sulfatide species in raft and non-raft membranes and has the potential to be applied to preparations from other tissues. Copyright © 2014 Elsevier Inc. All rights reserved.

Differential Association of the Na+/H+ Exchanger Regulatory Factor (NHERF) Family of Adaptor Proteins with the Raft-and the Non-Raft Brush Border Membrane Fractions of NHE3

PubMed Central

Sultan, Ayesha; Luo, Min; Yu, Qin; Riederer, Brigitte; Xia, Weiliang; Chen, Mingmin; Lissner, Simone; Gessner, Johannes E.; Donowitz, Mark; Yun, C. Chris; deJonge, Hugo; Lamprecht, Georg; Seidler, Ursula

2014-01-01

Background/Aims Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50), NHERF2 (E3KARP), and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM. Methods Detergent resistant membranes (“lipid rafts”) were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO2/HCO3− mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. Results NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. Conclusions The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs. PMID:24297041

Identification of AQP5 in lipid rafts and its translocation to apical membranes by activation of M3 mAChRs in interlobular ducts of rat parotid gland.

PubMed

Ishikawa, Yasuko; Yuan, Zhenfang; Inoue, Noriko; Skowronski, Mariusz T; Nakae, Yoshiko; Shono, Masayuki; Cho, Gota; Yasui, Masato; Agre, Peter; Nielsen, Søren

2005-11-01

Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. M(3) muscarinic acetylcholine receptor (mAChR)-induced changes in AQP5 localization in rat parotid glands were investigated with immunofluorescence or immunoelectron microscopy, detergent solubility, and gradient density floatation assays. Confocal microscopy revealed AQP5 localization in intracellular vesicles of interlobular duct cells in rat parotid glands and AQP5 trafficking to the APM 10 min after injection of the mAChR agonist cevimeline. Conversely, 60 min after injection, there was a diffuse pattern of AQP5 staining in the cell cytoplasm. The calcium ionophore A-23187 mimicked the effects of cevimeline. Immunoelectron microscopic studies confirmed that cevimeline induced AQP5 trafficking from intracellular structures to APMs in the interlobular duct cells of rat parotid glands. Lipid raft markers flotillin-2 and GM1 colocalized with AQP5 and moved with AQP5 in response to cevimeline. Under control conditions, the majority of AQP5 localized in the Triton X-100-insoluble fraction and floated to the light-density fraction on discontinuous density gradients. After 10-min incubation of parotid tissue slices with cevimeline or A-23187, AQP5 levels decreased in the Triton X-100-insoluble fraction and increased in the Triton X-100-soluble fraction. Thus AQP5 localizes in the intracellular lipid rafts, and M(3) mAChR activation induces AQP5 trafficking to the APM with lipid rafts via intracellular Ca(2+) signaling and induces AQP5 dissociation from lipid rafts to nonrafts on the APM in the interlobular duct cells of rat parotid glands.

Interdependence of laminin-mediated clustering of lipid rafts and the dystrophin complex in astrocytes.

PubMed

Noël, Geoffroy; Tham, Daniel Kai Long; Moukhles, Hakima

2009-07-17

Astrocyte endfeet surrounding blood vessels are active domains involved in water and potassium ion transport crucial to the maintenance of water and potassium ion homeostasis in brain. A growing body of evidence points to a role for dystroglycan and its interaction with perivascular laminin in the targeting of the dystrophin complex and the water-permeable channel, aquaporin 4 (AQP4), at astrocyte endfeet. However, the mechanisms underlying such compartmentalization remain poorly understood. In the present study we found that AQP4 resided in Triton X-100-insoluble fraction, whereas dystroglycan was recovered in the soluble fraction in astrocytes. Cholesterol depletion resulted in the translocation of a pool of AQP4 to the soluble fraction indicating that its distribution is indeed associated with cholesterol-rich membrane domains. Upon laminin treatment AQP4 and the dystrophin complex, including dystroglycan, reorganized into laminin-associated clusters enriched for the lipid raft markers GM1 and flotillin-1 but not caveolin-1. Reduced diffusion rates of GM1 in the laminin-induced clusters were indicative of the reorganization of raft components in these domains. In addition, both cholesterol depletion and dystroglycan silencing reduced the number and area of laminin-induced clusters of GM1, AQP4, and dystroglycan. These findings demonstrate the interdependence between laminin binding to dystroglycan and GM1-containing lipid raft reorganization and provide novel insight into the dystrophin complex regulation of AQP4 polarization in astrocytes.

Functional analysis of alpha5beta1 integrin and lipid rafts in invasion of epithelial cells by Porphyromonas gingivalis using fluorescent beads coated with bacterial membrane vesicles.

PubMed

Tsuda, Kayoko; Furuta, Nobumichi; Inaba, Hiroaki; Kawai, Shinji; Hanada, Kentaro; Yoshimori, Tamotsu; Amano, Atsuo

2008-01-01

Porphyromonas gingivalis, a periodontal pathogen, was previously suggested to exploit alpha5beta1 integrin and lipid rafts to invade host cells. However, it is unknown if the functional roles of these host components are distinct from one another during bacterial invasion. In the present study, we analyzed the mechanisms underlying P. gingivalis invasion, using fluorescent beads coated with bacterial membrane vesicles (MV beads). Cholesterol depletion reagents including methyl-beta-cyclodextrin (MbetaCD) drastically inhibited the entry of MV beads into epithelial cells, while they were less effective on bead adhesion to the cells. Bead entry was also abolished in CHO cells deficient in sphingolipids, components of lipid rafts, whereas adhesion was negligibly influenced. Following MbetaCD treatment, downstream events leading to actin polymerization were abolished; however, alpha5beta1 integrin was recruited to beads attached to the cell surface. Dominant-negative Rho GTPase Rac1 abolished cellular engulfment of the beads, whereas dominant-negative Cdc42 did not. Following cellular interaction with the beads, Rac1 was found to be translocated to the lipid rafts fraction, which was inhibited by MbetaCD. These results suggest that alpha5beta1 integrin, independent of lipid rafts, promotes P. gingivalis adhesion to epithelial cells, while the subsequent uptake process requires lipid raft components for actin organization, with Rho GTPase Rac1.

Disruption of lipid rafts interferes with the interaction of Toxoplasma gondii with macrophages and epithelial cells.

PubMed

Cruz, Karla Dias; Cruz, Thayana Araújo; Veras de Moraes, Gabriela; Paredes-Santos, Tatiana Christina; Attias, Marcia; de Souza, Wanderley

2014-01-01

The intracellular parasite Toxoplasma gondii can penetrate any warm-blooded animal cell. Conserved molecular assemblies of host cell plasma membranes should be involved in the parasite-host cell recognition. Lipid rafts are well-conserved membrane microdomains that contain high concentrations of cholesterol, sphingolipids, glycosylphosphatidylinositol, GPI-anchored proteins, and dually acylated proteins such as members of the Src family of tyrosine kinases. Disturbing lipid rafts of mouse peritoneal macrophages and epithelial cells of the lineage LLC-MK2 with methyl-beta cyclodextrin (M β CD) and filipin, which interfere with cholesterol or lidocaine, significantly inhibited internalization of T. gondii in both cell types, although adhesion remained unaffected in macrophages and decreased only in LLC-MK2 cells. Scanning and transmission electron microscopy confirmed these observations. Results are discussed in terms of the original role of macrophages as professional phagocytes versus the LLC-MK2 cell lineage originated from kidney epithelial cells.

Absence of correlation between oxysterol accumulation in lipid raft microdomains, calcium increase, and apoptosis induction on 158N murine oligodendrocytes.

PubMed

Ragot, Kévin; Mackrill, John J; Zarrouk, Amira; Nury, Thomas; Aires, Virginie; Jacquin, Agnès; Athias, Anne; Pais de Barros, Jean-Paul; Véjux, Anne; Riedinger, Jean-Marc; Delmas, Dominique; Lizard, Gérard

2013-07-01

There is some evidence that oxidized derivatives of cholesterol, 7-ketocholesterol (7KC) and 7β-hydroxycholesterol (7βOHC), are increased in the plasma of patients with neurodegenerative diseases associated with demyelinization of the central nervous system (CNS). It was therefore of interest to investigate the effects of these oxysterols on oligodendrocytes, the myelin-forming cells in the CNS. To this end, 158N murine oligodendrocytes were treated with 7KC or 7βOHC inducing an apoptotic mode of cell death characterized by condensation/fragmentation of the nuclei, dephosphorylation of Akt and GSK3, mitochondrial depolarization involving Mcl-1, and caspase-3 activation. In contrast, under treatment with 27-hydroxycholesterol (27OHC), no cell death was observed. When the cells were stained with Fura-2, no significant Ca(2+) rise was found with the different oxysterols, whereas strong signals were detected with ionomycin used as positive control. At concentrations which induced apoptosis, 7KC but not 7βOHC accumulated in lipid rafts. Although not cytotoxic, 27OHC was mainly detected in lipid rafts. It is noteworthy that α-tocopherol (but not ellagic acid and resveratrol) was able to counteract 7KC- and 7βOHC-induced apoptosis and to decrease the accumulation of 7KC and 27OHC in lipid rafts. Thus, in 158N cells, the ability of oxysterols to trigger a mode of cell death by apoptosis involving GSK-3 and caspase-3 activation is independent of the increase in the Ca(2+) level and of their accumulation in lipid raft microdomains. Copyright © 2013 Elsevier Inc. All rights reserved.

Lipid raft-mediated miR-3908 inhibition of migration of breast cancer cell line MCF-7 by regulating the interactions between AdipoR1 and Flotillin-1.

PubMed

Li, Yuan; Shan, Fei; Chen, Jinglong

2017-03-21

The mechanisms of lipid raft regulation by microRNAs in breast cancer are not fully understood. This work focused on the evaluation and identification of miR-3908, which may be a potential biomarker related to the migration of breast cancer cells, and elucidates lipid-raft-regulating cell migration in breast cancer. To confirm the prediction that miR-3908 is matched with AdipoR1, we used 3'-UTR luciferase activity of AdipoR1 to assess this. Then, human breast cancer cell line MCF-7 was cultured in the absence or presence of the mimics or inhibitors of miR-3908, after which the biological functions of MCF-7 cells were analyzed. The protein expression of AdipoR1, AMPK, and SIRT-1 were examined. The interaction between AdipoR1 and Flotillin-1, or its effects on lipid rafts on regulating cell migration of MCF-7, was also investigated. AdipoR1 is a direct target of miR-3908. miR-3908 suppresses the expression of AdipoR1 and its downstream pathway genes, including AMPK, p-AMPK, and SIRT-1. miR-3908 enhances the process of breast cancer cell clonogenicity. miR-3908 exerts its effects on the proliferation and migration of MCF-7 cells, which are mediated by lipid rafts regulating AdipoR1's ability to bind Flotillin-1. miR-3908 is a crucial mediator of the migration process in breast cancer cells. Lipid rafts regulate the interactions between AdipoR1 and Flotillin-1 and then the migration process associated with miR-3908 in MCF-7 cells. Our findings suggest that targeting miR-3908 and the lipid raft, may be a promising strategy for the treatment and prevention of breast cancer.

Expressed Glycosylphosphatidylinositol-Anchored Horseradish Peroxidase Identifies Co-Clustering Molecules in Individual Lipid Raft Domains

PubMed Central

Miyagawa-Yamaguchi, Arisa; Kotani, Norihiro; Honke, Koichi

2014-01-01

Lipid rafts that are enriched in glycosylphosphatidylinositol (GPI)-anchored proteins serve as a platform for important biological events. To elucidate the molecular mechanisms of these events, identification of co-clustering molecules in individual raft domains is required. Here we describe an approach to this issue using the recently developed method termed enzyme-mediated activation of radical source (EMARS), by which molecules in the vicinity within 300 nm from horseradish peroxidase (HRP) set on the probed molecule are labeled. GPI-anchored HRP fusion proteins (HRP-GPIs), in which the GPI attachment signals derived from human decay accelerating factor and Thy-1 were separately connected to the C-terminus of HRP, were expressed in HeLa S3 cells, and the EMARS reaction was catalyzed by these expressed HRP-GPIs under a living condition. As a result, these different HRP-GPIs had differences in glycosylation and localization and formed distinct clusters. This novel approach distinguished molecular clusters associated with individual GPI-anchored proteins, suggesting that it can identify co-clustering molecules in individual raft domains. PMID:24671047

CHOLINE PARTIALLY PREVENTS THE IMPACT OF ETHANOL ON THE LIPID RAFT DEPENDENT FUNCTIONS OF L1 CELL ADHESION MOLECULE

PubMed Central

Tang, Ningfeng; Bamford, Penny; Jones, Jace; He, Min; Kane, Maureen A.; Mooney, Sandra M.; Bearer, Cynthia F.

2014-01-01

Background Fetal Alcohol Spectrum Disorder, the leading known cause of mental retardation, is caused by alcohol exposure during pregnancy. One mechanism of ethanol teratogenicity is the disruption of the function of L1 cell adhesion molecule (L1). These functions include enhancement of neurite outgrowth, trafficking through lipid rafts, and signal transduction. Recent data have shown that choline supplementation of rat pups reduces the effects of ethanol on neurobehavior. We sought to determine if choline could prevent the effect of ethanol on L1 function using a simple experimental system. Methods Cerebellar granule neurons (CGN) from postnatal day 6 rat pups were cultured with and without supplemental choline, and the effects on L1 signaling, lipid raft distribution and neurite outgrowth were measured in the presence or absence of ethanol. Results Choline significantly reduced the effect of ethanol on L1 signaling, the distribution of L1 in lipid rafts and L1 mediated neurite outgrowth. However, choline supplemented ethanol exposed cultures remained significantly different than controls. Conclusions Choline pretreatment of CGN significantly reduces the disruption of L1 function by ethanol, but does not completely return L1 function to baseline. This experimental system will enable discovery of the mechanism of the neuroprotective effect of choline. PMID:25421509

Raft-Like Membrane Domains in Pathogenic Microorganisms

PubMed Central

Farnoud, Amir M.; Toledo, Alvaro M.; Konopka, James B.; Del Poeta, Maurizio; London, Erwin

2016-01-01

The lipid bilayer of the plasma membrane is thought to be compartmentalized by the presence of lipid-protein microdomains. In eukaryotic cells, microdomains composed of sterols and sphingolipids packed in a liquid-ordered state, commonly known as lipid rafts, are believed to exist. While less studied in bacterial cells, reports on the presence of sterol or protein-mediated microdomains in bacterial cell membranes are also appearing with increasing frequency. Recent efforts have been focused on addressing the biophysical and biochemical properties of lipid rafts. However, most studies have been focused on synthetic membranes, mammalian cells, and/or model, non-pathogenic microorganisms. Much less is known about microdomains in the plasma membrane of pathogenic microorganisms. This review attempts to provide an overview of the current state of knowledge of lipid rafts in pathogenic fungi and the developing field of microdomains in pathogenic bacteria. The current literature on the structure and function and of microdomains is reviewed and the potential role of microdomains in growth, pathogenesis, and drug resistance of pathogens are discussed. Better insight into the structure and function of membrane microdomains in pathogenic microorganisms might lead to a better understanding of the process of pathogenesis and development of raft-mediated approaches for new methods of therapy. PMID:26015285

The mystery of membrane organization: composition, regulation and physiological relevance of lipid rafts

PubMed Central

Sezgin, Erdinc; Levental, Ilya; Mayor, Satyajit; Eggeling, Christian

2017-01-01

Cellular plasma membranes are laterally heterogeneous, featuring a variety of distinct subcompartments that differ in their biophysical properties and composition. A large body of research has focused on understanding the basis for this heterogeneity and its physiological relevance. The membrane raft hypothesis formalized a physicochemical principle for a subtype of such lateral membrane heterogeneity, wherein the preferential associations of cholesterol and saturated lipids drives the formation of relatively packed (ordered) membrane domains that selectively recruit certain lipids and proteins. Recent years have yielded new insights into this concept and its in vivo relevance, primarily owing to the development of biochemical and biophysical technologies. PMID:28356571

Pathogens: raft hijackers.

PubMed

Mañes, Santos; del Real, Gustavo; Martínez-A, Carlos

2003-07-01

Throughout evolution, organisms have developed immune-surveillance networks to protect themselves from potential pathogens. At the cellular level, the signalling events that regulate these defensive responses take place in membrane rafts--dynamic microdomains that are enriched in cholesterol and glycosphingolipids--that facilitate many protein-protein and lipid-protein interactions at the cell surface. Pathogens have evolved many strategies to ensure their own survival and to evade the host immune system, in some cases by hijacking rafts. However, understanding the means by which pathogens exploit rafts might lead to new therapeutic strategies to prevent or alleviate certain infectious diseases, such as those caused by HIV-1 or Ebola virus.

Sorafenib suppresses TGF-β responses by inducing caveolae/lipid raft-mediated internalization/degradation of cell-surface type II TGF-β receptors: Implications in development of effective adjunctive therapy for hepatocellular carcinoma.

PubMed

Chung, Chih-Ling; Wang, Shih-Wei; Sun, Wei-Chih; Shu, Chih-Wen; Kao, Yu-Chen; Shiao, Meng-Shin; Chen, Chun-Lin

2018-04-18

Sorafenib is the only FDA approved drug for the treatment of advanced hepatocellular carcinoma (HCC) and other malignancies. Studies indicate that TGF-β signalling is associated with tumour progression in HCC. Autocrine and paracrine TGF-β promotes tumour growth and malignancy by inducing epithelial-mesenchymal transition (EMT). Sorafenib is believed to antagonize tumour progression by inhibiting TGF-β-induced EMT. It improves survival of patients but HCC later develops resistance and relapses. The underlying mechanism of resistance is unknown. Understanding of the molecular mechanism of sorafenib inhibition of TGF-β-induced signalling or responses in HCC may lead to development of adjunctive effective therapy for HCC. In this study, we demonstrate that sorafenib suppresses TGF-β responsiveness in hepatoma cells, hepatocytes, and animal liver, mainly by downregulating cell-surface type II TGF-β receptors (TβRII) localized in caveolae/lipid rafts and non-lipid raft microdomains via caveolae/lipid rafts-mediated internalization and degradation. Furthermore, sorafenib-induced downregulation and degradation of cell-surface TβRII is prevented by simultaneous treatment with a caveolae disruptor or lysosomal inhibitors. On the other hand, sorafenib only downregulates cell-surface TβRII localized in caveolae/lipid rafts but not localized in non-lipid raft microdomains in hepatic stellate cells. These results suggest that sorafenib inhibits TGF-β signalling mainly by inducing caveolae/lipid raft-mediated internalization and degradation of cell-surface TβR-II in target cells. They may also imply that treatment with agents which promote formation of caveolae/lipid rafts, TGF-β receptor kinase inhibitors (e.g., LY2157299) or TGF-β peptide antagonists (by liver-targeting delivery) may be considered as effective adjunct therapy with sorafenib for HCC. Copyright © 2018 Elsevier Inc. All rights reserved.

On scattered waves and lipid domains: detecting membrane rafts with X-rays and neutrons

DOE PAGES

Marquardt, Drew; Heberle, Frederick A.; Nickels, Jonathan D.; ...

2015-09-21

In order to understand the biological role of lipids in cell membranes, it is necessary to determine the mesoscopic structure of well-defined model membrane systems. Neutron and X-ray scattering are non-invasive, probe-free techniques that have been used extensively in such systems to probe length scales ranging from angstroms to microns, and dynamics occurring over picosecond to millisecond time scales. Finally, recent developments in the area of phase separated lipid systems mimicking membrane rafts will be presented, and the underlying concepts of the different scattering techniques used to study them will be discussed in detail.

Lipid Rafts Act as Specialized Domains for Tetanus Toxin Binding and Internalization into Neurons

PubMed Central

Herreros, Judit; Ng, Tony; Schiavo, Giampietro

2001-01-01

Tetanus (TeNT) is a zinc protease that blocks neurotransmission by cleaving the synaptic protein vesicle-associated membrane protein/synaptobrevin. Although its intracellular catalytic activity is well established, the mechanism by which this neurotoxin interacts with the neuronal surface is not known. In this study, we characterize p15s, the first plasma membrane TeNT binding proteins and we show that they are glycosylphosphatidylinositol-anchored glycoproteins in nerve growth factor (NGF)-differentiated PC12 cells, spinal cord cells, and purified motor neurons. We identify p15 as neuronal Thy-1 in NGF-differentiated PC12 cells. Fluorescence lifetime imaging microscopy measurements confirm the close association of the binding domain of TeNT and Thy-1 at the plasma membrane. We find that TeNT is recruited to detergent-insoluble lipid microdomains on the surface of neuronal cells. Finally, we show that cholesterol depletion affects a raft subpool and blocks the internalization and intracellular activity of the toxin. Our results indicate that TeNT interacts with target cells by binding to lipid rafts and that cholesterol is required for TeNT internalization and/or trafficking in neurons. PMID:11598183

Ylpex5 mutation partially suppresses the defective hyphal growth of a Yarrowia lipolytica ceramide synthase mutant, Yllac1, by recovering lipid raft polarization and vacuole morphogenesis.

PubMed

Bal, Jyotiranjan; Lee, Hye-Jeong; Cheon, Seon Ah; Lee, Kyung Jin; Oh, Doo-Byoung; Kim, Jeong-Yoon

2013-01-01

Sphingolipids are involved in cell differentiation and morphogenesis in eukaryotic cells. In this study, YlLac1p, a ceramide synthase required for glucosylceramide (GlcCer) synthesis, was found to be essential for hyphal growth in Yarrowia lipolytica. Y. lipolytica GlcCer was shown to be composed of a C16:0 fatty acid, which is hydroxylated at C2, and a C18:2 long chain base, which is unsaturated at both C4 and C8 and methylated at C9. Domain swapping analysis revealed that the entire TRAM/Lag1/CLN8 (TLC) domain, not the Lag1 motif, is crucial for the function of YlLac1p. YlDes1p, the C4 desaturase of the ceramide synthesized by YlLac1p, was also required for Y. lipolytica morphogenesis. Both Yllac1Δ and Yldes1Δ mutants neither polarize lipid rafts nor form normal vacuoles. Interestingly, mutation in YlPEX5, which encode a peroxisomal targeting signal receptor, partially suppressed the defective hyphal growth of Yllac1Δ. The Yllac1ΔYlpex5Δ mutant restored the ability to polarize lipid rafts and to form normal vacuoles, although it could not synthesize GlcCer. Taken together, our results suggest that GlcCer or GlcCer derivatives may be involved in hyphal morphogenesis in Y. lipolytica, at least in part, by affecting polarization of lipid rafts and vacuole morphogenesis. Copyright © 2012 Elsevier Inc. All rights reserved.

Drug Uptake, Lipid Rafts, and Vesicle Trafficking Modulate Resistance to an Anticancer Lysophosphatidylcholine Analogue in Yeast*

PubMed Central

Cuesta-Marbán, Álvaro; Botet, Javier; Czyz, Ola; Cacharro, Luis M.; Gajate, Consuelo; Hornillos, Valentín; Delgado, Javier; Zhang, Hui; Amat-Guerri, Francisco; Acuña, A. Ulises; McMaster, Christopher R.; Revuelta, José Luis; Zaremberg, Vanina; Mollinedo, Faustino

2013-01-01

The ether-phospholipid edelfosine, a prototype antitumor lipid (ATL), kills yeast cells and selectively kills several cancer cell types. To gain insight into its mechanism of action, we performed chemogenomic screens in the Saccharomyces cerevisiae gene-deletion strain collection, identifying edelfosine-resistant mutants. LEM3, AGP2, and DOC1 genes were required for drug uptake. Edelfosine displaced the essential proton pump Pma1p from rafts, inducing its internalization into the vacuole. Additional ATLs, including miltefosine and perifosine, also displaced Pma1p from rafts to the vacuole, suggesting that this process is a major hallmark of ATL cytotoxicity in yeast. Radioactive and synthetic fluorescent edelfosine analogues accumulated in yeast plasma membrane rafts and subsequently the endoplasmic reticulum. Although both edelfosine and Pma1p were initially located at membrane rafts, internalization of the drug toward endoplasmic reticulum and Pma1p to the vacuole followed different routes. Drug internalization was not dependent on endocytosis and was not critical for yeast cytotoxicity. However, mutants affecting endocytosis, vesicle sorting, or trafficking to the vacuole, including the retromer and ESCRT complexes, prevented Pma1p internalization and were edelfosine-resistant. Our data suggest that edelfosine-induced cytotoxicity involves raft reorganization and retromer- and ESCRT-mediated vesicular transport and degradation of essential raft proteins leading to cell death. Cytotoxicity of ATLs is mainly dependent on the changes they induce in plasma membrane raft-located proteins that lead to their internalization and subsequent degradation. Edelfosine toxicity can be circumvented by inactivating genes that then result in the recycling of internalized cell-surface proteins back to the plasma membrane. PMID:23335509

The Ia.2 Epitope Defines a Subset of Lipid Raft Resident MHC Class II Molecules Crucial to Effective Antigen Presentation1

PubMed Central

Busman-Sahay, Kathleen; Sargent, Elizabeth; Harton, Jonathan A.; Drake, James R.

2016-01-01

Previous work has established that binding of the 11-5.2 anti-I-Ak mAb, which recognizes the Ia.2 epitope on I-Ak class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-Ak mAb that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-Ak molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2 bearing subset of I-Ak class II molecules is critically necessary for effective B cell–T cell interactions especially at low antigen doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-Ak class II molecules possessing a β chain-tethered HEL peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2 negative tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous antigen to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II confomer vital to the initiation of MHC class II restricted B cell–T cell interactions. PMID:21543648

Raft-like membrane domains in pathogenic microorganisms.

PubMed

Farnoud, Amir M; Toledo, Alvaro M; Konopka, James B; Del Poeta, Maurizio; London, Erwin

2015-01-01

The lipid bilayer of the plasma membrane is thought to be compartmentalized by the presence of lipid-protein microdomains. In eukaryotic cells, microdomains composed of sterols and sphingolipids, commonly known as lipid rafts, are believed to exist, and reports on the presence of sterol- or protein-mediated microdomains in bacterial cell membranes are also appearing. Despite increasing attention, little is known about microdomains in the plasma membrane of pathogenic microorganisms. This review attempts to provide an overview of the current state of knowledge of lipid rafts in pathogenic fungi and bacteria. The current literature on characterization of microdomains in pathogens is reviewed, and their potential role in growth, pathogenesis, and drug resistance is discussed. Better insight into the structure and function of membrane microdomains in pathogenic microorganisms might lead to a better understanding of their pathogenesis and development of raft-mediated approaches for therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

Choline partially prevents the impact of ethanol on the lipid raft dependent functions of l1 cell adhesion molecule.

PubMed

Tang, Ningfeng; Bamford, Penny; Jones, Jace; He, Min; Kane, Maureen A; Mooney, Sandra M; Bearer, Cynthia F

2014-11-01

Fetal alcohol spectrum disorder, the leading known cause of mental retardation, is caused by alcohol exposure during pregnancy. One mechanism of ethanol (EtOH) teratogenicity is the disruption of the functions of L1 cell adhesion molecule (L1). These functions include enhancement of neurite outgrowth, trafficking through lipid rafts, and signal transduction. Recent data have shown that choline supplementation of rat pups reduces the effects of EtOH on neurobehavior. We sought to determine whether choline could prevent the effect of EtOH on L1 function using a simple experimental system. Cerebellar granule neurons (CGN) from postnatal day 6 rat pups were cultured with and without supplemental choline, and the effects on L1 signaling, lipid raft distribution, and neurite outgrowth were measured in the presence or absence of EtOH. Choline significantly reduced the effect of EtOH on L1 signaling, the distribution of L1 in lipid rafts and L1-mediated neurite outgrowth. However, choline supplemented EtOH-exposed cultures remained significantly different than controls. Choline pretreatment of CGN significantly reduces the disruption of L1 function by EtOH, but does not completely return L1 function to baseline. This experimental system will enable discovery of the mechanism of the neuroprotective effect of choline. Copyright © 2014 by the Research Society on Alcoholism.

Regulation of raft-dependent endocytosis

PubMed Central

Lajoie, P; Nabi, IR

2007-01-01

Abstract Raft-dependent endocytosis is in large part defined as the cholesterol-sensitive, clathrin-independent internalization of ligands and receptors from the plasma membrane. It encompasses the endocytosis of caveo-lae, smooth plasmalemmal vesicles that form a subdomain of cholesterol and sphingolipid-rich lipid rafts and that are enriched for caveolin-1. While sharing common mechanisms, like cholesterol sensitivity, raft endocytic routes show differential regulation by various cellular components including caveolin-1, dynamin-2 and regulators of the actin cytoskeleton. Dynamin-dependent raft pathways, mediated by caveolae and morphologically equivalent non-caveolin vesicular intermediates, are referred to as caveolae/raft-dependent endocytosis. In contrast, dynamin-independent raft pathways are mediated by non-caveolar intermediates. Raft-dependent endocytosis is regulated by tyrosine kinase inhibitors and, through the regulation of the internalization of various ligands, receptors and effectors, is also a determinant of cellular signaling. In this review, we characterize and discuss the regulation of raft-dependent endocytic pathways and the role of key regulators such as caveolin-1. PMID:17760830

Neuroglobin overexpression plays a pivotal role in neuroprotection through mitochondrial raft-like microdomains in neuroblastoma SK-N-BE2 cells.

PubMed

Garofalo, Tina; Ferri, Alberto; Sorice, Maurizio; Azmoon, Pardis; Grasso, Maria; Mattei, Vincenzo; Capozzi, Antonella; Manganelli, Valeria; Misasi, Roberta

2018-04-01

Since stressing conditions induce a relocalization of endogenous human neuroglobin (NGB) to mitochondria, this research is aimed to evaluate the protective role of NGB overexpression against neurotoxic stimuli, through mitochondrial lipid raft-associated complexes. To this purpose, we built a neuronal model of oxidative stress by the use of human dopaminergic neuroblastoma cells, SK-N-BE2, stably overexpressing NGB by transfection and treated with 1-methyl-4-phenylpyridinium ion (MPP+). We preliminary observed the redistribution of NGB to mitochondria following MPP+ treatment. The analysis of mitochondrial raft-like microdomains revealed that, following MPP+ treatment, NGB translocated to raft fractions (Triton X-100-insoluble), where it interacts with ganglioside GD3. Interestingly, the administration of agents capable of perturbating microdomain before MPP+ treatment, significantly affected viability in SK-N-BE2-NGB cells. The overexpression of NGB was able to abrogate the mitochondrial injuries on complex IV activity or mitochondrial morphology induced by MPP+ administration. The protective action of NGB on mitochondria only takes place if the mitochondrial lipid(s) rafts-like microdomains are intact, indeed NGB fails to protect complex IV activity when purified mitochondria were treated with the lipid rafts disruptor methyl-β-cyclodextrin. Thus, our unique in vitro model of stably transfected cells overexpressing endogenous NGB allowed us to suggest that the role in neuroprotection played by NGB is reliable only through interaction with mitochondrial lipid raft-associated complexes. Copyright © 2018 Elsevier Inc. All rights reserved.

Raft-based sphingomyelin interactions revealed by new fluorescent sphingomyelin analogs

PubMed Central

Kinoshita, Masanao; Suzuki, Kenichi G.N.; Takada, Misa; Ano, Hikaru; Abe, Mitsuhiro; Makino, Asami; Kobayashi, Toshihide; Hirosawa, Koichiro M.; Fujiwara, Takahiro K.; Murata, Michio

2017-01-01

Sphingomyelin (SM) has been proposed to form cholesterol-dependent raft domains and sphingolipid domains in the plasma membrane (PM). How SM contributes to the formation and function of these domains remains unknown, primarily because of the scarcity of suitable fluorescent SM analogs. We developed new fluorescent SM analogs by conjugating a hydrophilic fluorophore to the SM choline headgroup without eliminating its positive charge, via a hydrophilic nonaethylene glycol linker. The new analogs behaved similarly to the native SM in terms of their partitioning behaviors in artificial liquid order-disorder phase-separated membranes and detergent-resistant PM preparations. Single fluorescent molecule tracking in the live-cell PM revealed that they indirectly interact with each other in cholesterol- and sphingosine backbone–dependent manners, and that, for ∼10–50 ms, they undergo transient colocalization-codiffusion with a glycosylphosphatidylinositol (GPI)-anchored protein, CD59 (in monomers, transient-dimer rafts, and clusters), in CD59-oligomer size–, cholesterol-, and GPI anchoring–dependent manners. These results suggest that SM continually and rapidly exchanges between CD59-associated raft domains and the bulk PM. PMID:28330937

Dual acylation and lipid raft association of Src-family protein tyrosine kinases are required for SDF-1/CXCL12-mediated chemotaxis in the Jurkat human T cell lymphoma cell line.

PubMed

Zaman, Sabiha N; Resek, Mary E; Robbins, Stephen M

2008-10-01

Chemokines play pivotal roles in regulating a wide variety of biological processes by modulating cell migration and recruitment. Deregulation of chemokine signaling can alter cell recruitment, contributing to the pathogenic states associated with autoimmune disease, inflammatory disorders, and sepsis. During chemotaxis, lipid rafts and their resident signaling molecules have been demonstrated to partition to different parts of the cell. Herein, we investigated the role of lipid raft resident Src-family kinases (SFK) in stromal cell-derived factor 1/CXCL12-mediated chemotaxis. We have shown that Lck-deficient J.CaM 1.6 cells are defective in CXCL12-mediated chemotaxis in contrast to their parental counterpart, Jurkat cells. Ectopic expression of the SFK hematopoietic cell kinase (Hck) in J.CaM 1.6 cells reconstituted CXCL12 responsiveness. The requirement of lipid raft association of SFK was assessed using both isoforms of Hck: the dually acylated p59(Hck) isoform that is targeted to lipid rafts and the monoacylated p61(Hck) isoform that is nonraft-associated. We have shown using several gain and loss of acylation alleles that dual acylation of Hck was required for CXCL12-mediated chemotaxis in J.CaM 1.6 cells. These results highlight the importance of the unique microenvironment provided by lipid rafts and their specific contribution in providing specificity to CXCL12 signaling.

Continuity of Monolayer-Bilayer Junctions for Localization of Lipid Raft Microdomains in Model Membranes

PubMed Central

Ryu, Yong-Sang; Wittenberg, Nathan J.; Suh, Jeng-Hun; Lee, Sang-Wook; Sohn, Youngjoo; Oh, Sang-Hyun; Parikh, Atul N.; Lee, Sin-Doo

2016-01-01

We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed between the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates. PMID:27230411

Improved platelet survival after cold storage by prevention of glycoprotein Ibα clustering in lipid rafts

PubMed Central

Gitz, Eelo; Koekman, Cornelis A; van den Heuvel, Dave J.; Deckmyn, Hans; Akkerman, Jan W.; Gerritsen, Hans C.; Urbanus, Rolf T.

2012-01-01

Background Storing platelets for transfusion at room temperature increases the risk of microbial infection and decreases platelet functionality, leading to out-date discard rates of up to 20%. Cold storage may be a better alternative, but this treatment leads to rapid platelet clearance after transfusion, initiated by changes in glycoprotein Ibα, the receptor for von Willebrand factor. Design and Methods: We examined the change in glycoprotein Ibα distribution using Förster resonance energy transfer by time-gated fluorescence lifetime imaging microscopy. Results Cold storage induced deglycosylation of glycoprotein Ibα ectodomain, exposing N-acetyl-Dglucosamine residues, which sequestered with GM1 gangliosides in lipid rafts. Raft-associated glycoprotein Ibα formed clusters upon binding of 14-3-3ζ adaptor proteins to its cytoplasmic tail, a process accompanied by mitochondrial injury and phosphatidyl serine exposure. Cold storage left glycoprotein Ibα surface expression unchanged and although glycoprotein V decreased, the fall did not affect glycoprotein Ibα clustering. Prevention of glycoprotein Ibα clustering by blockade of deglycosylation and 14-3-3ζ translocation increased the survival of cold-stored platelets to above the levels of platelets stored at room temperature without compromising hemostatic functions. Conclusions We conclude that glycoprotein Ibα translocates to lipid rafts upon cold-induced deglycosylation and forms clusters by associating with 14-3-3ζ. Interference with these steps provides a means to enable cold storage of platelet concentrates in the near future. PMID:22733027

Lovastatin inhibits amyloid precursor protein (APP) beta-cleavage through reduction of APP distribution in Lubrol WX extractable low density lipid rafts.

PubMed

Won, Je-Seong; Im, Yeong-Bin; Khan, Mushfiquddin; Contreras, Miguel; Singh, Avtar K; Singh, Inderjit

2008-05-01

Previous studies have described that statins (inhibitors of cholesterol and isoprenoid biosynthesis) inhibit the output of amyloid-beta (Abeta) in the animal model and thus decrease risk of Alzheimer's disease. However, their action mechanism(s) in Abeta precursor protein (APP) processing and Abeta generation is not fully understood. In this study, we report that lovastatin treatment reduced Abeta output in cultured hippocampal neurons as a result of reduced APP levels and beta-secretase activities in low density Lubrol WX (non-ionic detergent) extractable lipid rafts (LDLR). Rather than altering cholesterol levels in lipid raft fractions and thus disrupting lipid raft structure, lovastatin decreased Abeta generation through down-regulating geranylgeranyl-pyrophosphate dependent endocytosis pathway. The inhibition of APP endocytosis by treatment with lovastatin and reduction of APP levels in LDLR fractions by treatment with phenylarsine oxide (a general endocytosis inhibitor) support the involvement of APP endocytosis in APP distribution in LDLR fractions and subsequent APP beta-cleavage. Moreover, lovastatin-mediated down-regulation of endocytosis regulators, such as early endosomal antigen 1, dynamin-1, and phosphatidylinositol 3-kinase activity, indicates that lovastatin modulates APP endocytosis possibly through its pleiotropic effects on endocytic regulators. Collectively, these data report that lovastatin mediates inhibition of LDLR distribution and beta-cleavage of APP in a geranylgeranyl-pyrophosphate and endocytosis-dependent manner.

LOVASTATIN INHIBITS AMYLOID PRECURSOR PROTEIN (APP) β-CLEAVAGE THROUGH REDUCTION OF APP DISTRIBUTION IN LUBROL WX EXTRACTABLE LOW DENSITY LIPID RAFTS

PubMed Central

Won, Je-Seong; Im, Yeong-Bin; Khan, Mushfiquddin; Contreras, Miguel; Singh, Avtar K.; Singh, Inderjit

2009-01-01

Previous studies have described that statins (inhibitors of cholesterol and isoprenoid biosynthesis) inhibit the output of amyloid-β (Aβ) in the animal model and thus decrease risk of Alzheimer's disease. However, their action mechanism(s) in APP processing and Aβ generation is not fully understood. Here we report that lovastatin treatment reduced Aβ output in cultured hippocampal neurons as a result of reduced Aβ precursor protein (APP) levels and β-secretase activities in low density Lubrol WX (non-ionic detergent) extractable lipid rafts (LDLR). Rather than altering cholesterol levels in lipid raft fractions and thus disrupting lipid raft structure, lovastatin decreased Aβ generation through down-regulating geranylgeranyl-pyrophosphate (GGPP) dependent endocytosis pathway. The inhibition of APP endocytosis by treatment with lovastatin and reduction of APP levels in LDLR fractions by treatment with phenylarsine oxide (a general endocytosis inhibitor) support the involvement of APP endocytosis in APP distribution in LDLR fractions and subsequent APP β-cleavage. Moreover, lovastatin-mediated down-regulation of endocytosis regulators, such as EEA1, dynamin-I and phosphatidylinositol-3 kinase activity, indicates that lovastatin modulates APP endocytosis possibly through its pleiotropic effects on endocytic regulators. Collectively, these data report that lovastatin mediates inhibition of LDLR distribution and β-cleavage of APP in a GGPP and endocytosis dependent manner. PMID:18266936

Recombinant VSV G proteins reveal a novel raft-dependent endocytic pathway in resorbing osteoclasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mulari, Mika T.K.; Centre for Military Medicine, Research Department, Lahti; Nars, Martin

2008-05-01

Transcytotic membrane flow delivers degraded bone fragments from the ruffled border to the functional secretory domain, FSD, in bone resorbing osteoclasts. Here we show that there is also a FSD-to-ruffled border trafficking pathway that compensates for the membrane loss during the matrix uptake process and that rafts are essential for this ruffled border-targeted endosomal pathway. Replacing the cytoplasmic tail of the vesicular stomatitis virus G protein with that of CD4 resulted in partial insolubility in Triton X-100 and retargeting from the peripheral non-bone facing plasma membrane to the FSD. Recombinant G proteins were subsequently endosytosed and delivered from the FSDmore » to the peripheral fusion zone of the ruffled border, which were both rich in lipid rafts as suggested by viral protein transport analysis and visualizing the rafts with fluorescent recombinant cholera toxin. Cholesterol depletion by methyl-{beta}-cyclodextrin impaired the ruffled border-targeted vesicle trafficking pathway and inhibited bone resorption dose-dependently as quantified by measuring the CTX and TRACP 5b secreted to the culture medium and by measuring the resorbed area visualized with a bi-phasic labeling method using sulpho-NHS-biotin and WGA-lectin. Thus, rafts are vital for membrane recycling from the FSD to the late endosomal/lysosomal ruffled border and bone resorption.« less

Myosin 1g Contributes to CD44 Adhesion Protein and Lipid Rafts Recycling and Controls CD44 Capping and Cell Migration in B Lymphocytes

PubMed Central

López-Ortega, Orestes; Santos-Argumedo, Leopoldo

2017-01-01

Cell migration and adhesion are critical for immune system function and involve many proteins, which must be continuously transported and recycled in the cell. Recycling of adhesion molecules requires the participation of several proteins, including actin, tubulin, and GTPases, and of membrane components such as sphingolipids and cholesterol. However, roles of actin motor proteins in adhesion molecule recycling are poorly understood. In this study, we identified myosin 1g as one of the important motor proteins that drives recycling of the adhesion protein CD44 in B lymphocytes. We demonstrate that the lack of Myo1g decreases the cell-surface levels of CD44 and of the lipid raft surrogate GM1. In cells depleted of Myo1g, the recycling of CD44 was delayed, the delay seems to be caused at the level of formation of recycling complex and entry into recycling endosomes. Moreover, a defective lipid raft recycling in Myo1g-deficient cells had an impact both on the capping of CD44 and on cell migration. Both processes required the transportation of lipid rafts to the cell surface to deliver signaling components. Furthermore, the extramembrane was essential for cell expansion and remodeling of the plasma membrane topology. Therefore, Myo1g is important during the recycling of lipid rafts to the membrane and to the accompanied proteins that regulate plasma membrane plasticity. Thus, Myosin 1g contributes to cell adhesion and cell migration through CD44 recycling in B lymphocytes. PMID:29321775

Chronic treatment with escitalopram but not R-citalopram translocates Galpha(s) from lipid raft domains and potentiates adenylyl cyclase: a 5-hydroxytryptamine transporter-independent action of this antidepressant compound.

PubMed

Zhang, Lanqiu; Rasenick, Mark M

2010-03-01

Chronic antidepressant treatment has been shown to increase adenylyl cyclase activity, in part, due to translocation of Galpha(s) from lipid rafts to a nonraft fraction of the plasma membrane where they engage in a more facile stimulation of adenylyl cyclase. This effect holds for multiple classes of antidepressants, and for serotonin uptake inhibitors, it occurs in the absence of the serotonin transporter. In the present study, we examined the change in the amount of Galpha(s) in lipid raft and whole cell lysate after exposing C6 cells to escitalopram. The results showed that chronic (but not acute) escitalopram decreased the content of Galpha(s) in lipid rafts, whereas there was no change in overall Galpha(s) content. These effects were drug dose- and exposure time-dependent. Although R-citalopram has been reported to antagonize some effects of escitalopram, this compound was without effect on Galpha(s) localization in lipid rafts, and R-citalopram did not inhibit these actions of escitalopram. Escitalopram treatment increased cAMP accumulation, and this seemed due to increased coupling between Galpha(s) and adenylyl cyclase. Thus, escitalopram is potent, rapid and efficacious in translocating Galpha(s) from lipid rafts, and this effect seems to occur independently of 5-hydroxytryptamine transporters. Our results suggest that, although antidepressants display distinct affinities for well identified targets (e.g., monoamine transporters), several presynaptic and postsynaptic molecules are probably modified during chronic antidepressant treatment, and these additional targets may be required for clinical efficacy of these drugs.

Chronic Treatment with Escitalopram but Not R-Citalopram Translocates Gαs from Lipid Raft Domains and Potentiates Adenylyl Cyclase: A 5-Hydroxytryptamine Transporter-Independent Action of This Antidepressant Compound

PubMed Central

Zhang, Lanqiu

2010-01-01

Chronic antidepressant treatment has been shown to increase adenylyl cyclase activity, in part, due to translocation of Gαs from lipid rafts to a nonraft fraction of the plasma membrane where they engage in a more facile stimulation of adenylyl cyclase. This effect holds for multiple classes of antidepressants, and for serotonin uptake inhibitors, it occurs in the absence of the serotonin transporter. In the present study, we examined the change in the amount of Gαs in lipid raft and whole cell lysate after exposing C6 cells to escitalopram. The results showed that chronic (but not acute) escitalopram decreased the content of Gαs in lipid rafts, whereas there was no change in overall Gαs content. These effects were drug dose- and exposure time-dependent. Although R-citalopram has been reported to antagonize some effects of escitalopram, this compound was without effect on Gαs localization in lipid rafts, and R-citalopram did not inhibit these actions of escitalopram. Escitalopram treatment increased cAMP accumulation, and this seemed due to increased coupling between Gαs and adenylyl cyclase. Thus, escitalopram is potent, rapid and efficacious in translocating Gαs from lipid rafts, and this effect seems to occur independently of 5-hydroxytryptamine transporters. Our results suggest that, although antidepressants display distinct affinities for well identified targets (e.g., monoamine transporters), several presynaptic and postsynaptic molecules are probably modified during chronic antidepressant treatment, and these additional targets may be required for clinical efficacy of these drugs. PMID:19996298

Measuring the strength of interaction between the Ebola fusion peptide and lipid rafts: implications for membrane fusion and virus infection.

PubMed

Freitas, Mônica S; Follmer, Cristian; Costa, Lilian T; Vilani, Cecília; Bianconi, M Lucia; Achete, Carlos Alberto; Silva, Jerson L

2011-01-13

The Ebola fusion peptide (EBO₁₆) is a hydrophobic domain that belongs to the GP2 membrane fusion protein of the Ebola virus. It adopts a helical structure in the presence of mimetic membranes that is stabilized by the presence of an aromatic-aromatic interaction established by Trp8 and Phe12. In spite of its infectious cycle becoming better understood recently, several steps still remain unclear, a lacuna that makes it difficult to develop strategies to block infection. In order to gain insight into the mechanism of membrane fusion, we probed the structure, function and energetics of EBO₁₆ and its mutant W8A, in the absence or presence of different lipid membranes, including isolated domain-resistant membranes (DRM), a good experimental model for lipid rafts. The depletion of cholesterol from living mammalian cells reduced the ability of EBO₁₆ to induce lipid mixing. On the other hand, EBO₁₆ was structurally sensitive to interaction with lipid rafts (DRMs), but the same was not observed for W8A mutant. In agreement with these data, W8A showed a poor ability to promote membrane aggregation in comparison to EBO₁₆. Single molecule AFM experiments showed a high affinity force pattern for the interaction of EBO₁₆ and DRM, which seems to be a complex energetic event as observed by the calorimetric profile. Our study is the first to show a strong correlation between the initial step of Ebola virus infection and cholesterol, thus providing a rationale for Ebola virus proteins being co-localized with lipid-raft domains. In all, the results show how small fusion peptide sequences have evolved to adopt highly specific and strong interactions with membrane domains. Such features suggest these processes are excellent targets for therapeutic and vaccine approaches to viral diseases.

The Relationship between Fenestrations, Sieve Plates and Rafts in Liver Sinusoidal Endothelial Cells

PubMed Central

McNerney, Gregory P.; Owen, Dylan M.; Zencak, Dusan; Zykova, Svetlana N.; Crane, Harry; Huser, Thomas; Quinn, Ronald J.; Smedsrød, Bård; Le Couteur, David G.; Cogger, Victoria C.

2012-01-01

Fenestrations are transcellular pores in endothelial cells that facilitate transfer of substrates between blood and the extravascular compartment. In order to understand the regulation and formation of fenestrations, the relationship between membrane rafts and fenestrations was investigated in liver sinusoidal endothelial cells where fenestrations are grouped into sieve plates. Three dimensional structured illumination microscopy, scanning electron microscopy, internal reflectance fluorescence microscopy and two-photon fluorescence microscopy were used to study liver sinusoidal endothelial cells isolated from mice. There was an inverse distribution between sieve plates and membrane rafts visualized by structured illumination microscopy and the fluorescent raft stain, Bodipy FL C5 ganglioside GM1. 7-ketocholesterol and/or cytochalasin D increased both fenestrations and lipid-disordered membrane, while Triton X-100 decreased both fenestrations and lipid-disordered membrane. The effects of cytochalasin D on fenestrations were abrogated by co-administration of Triton X-100, suggesting that actin disruption increases fenestrations by its effects on membrane rafts. Vascular endothelial growth factor (VEGF) depleted lipid-ordered membrane and increased fenestrations. The results are consistent with a sieve-raft interaction, where fenestrations form in non-raft lipid-disordered regions of endothelial cells once the membrane-stabilizing effects of actin cytoskeleton and membrane rafts are diminished. PMID:23029409

Continuity of monolayer-bilayer junctions for localization of lipid raft microdomains in model membranes

DOE PAGES

Ryu, Yong -Sang; Wittenberg, Nathan J.; Suh, Jeng -Hun; ...

2016-05-27

We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed betweenmore » the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Furthermore, our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates.« less

Exosome uptake depends on ERK1/2-heat shock protein 27 signaling and lipid Raft-mediated endocytosis negatively regulated by caveolin-1.

PubMed

Svensson, Katrin J; Christianson, Helena C; Wittrup, Anders; Bourseau-Guilmain, Erika; Lindqvist, Eva; Svensson, Lena M; Mörgelin, Matthias; Belting, Mattias

2013-06-14

The role of exosomes in cancer can be inferred from the observation that they transfer tumor cell derived genetic material and signaling proteins, resulting in e.g. increased tumor angiogenesis and metastasis. However, the membrane transport mechanisms and the signaling events involved in the uptake of these virus-like particles remain ill-defined. We now report that internalization of exosomes derived from glioblastoma (GBM) cells involves nonclassical, lipid raft-dependent endocytosis. Importantly, we show that the lipid raft-associated protein caveolin-1 (CAV1), in analogy with its previously described role in virus uptake, negatively regulates the uptake of exosomes. We find that exosomes induce the phosphorylation of several downstream targets known to associate with lipid rafts as signaling and sorting platforms, such as extracellular signal-regulated kinase-1/2 (ERK1/2) and heat shock protein 27 (HSP27). Interestingly, exosome uptake appears dependent on unperturbed ERK1/2-HSP27 signaling, and ERK1/2 phosphorylation is under negative influence by CAV1 during internalization of exosomes. These findings significantly advance our general understanding of exosome-mediated uptake and offer potential strategies for how this pathway may be targeted through modulation of CAV1 expression and ERK1/2 signaling.

Ethanol effects on binary and ternary supported lipid bilayers with gel/fluid domains and lipid rafts.

PubMed

Marquês, Joaquim T; Viana, Ana S; De Almeida, Rodrigo F M

2011-01-01

Ethanol-lipid bilayer interactions have been a recurrent theme in membrane biophysics, due to their contribution to the understanding of membrane structure and dynamics. The main purpose of this study was to assess the interplay between membrane lateral heterogeneity and ethanol effects. This was achieved by in situ atomic force microscopy, following the changes induced by sequential ethanol additions on supported lipid bilayers formed in the absence of alcohol. Binary phospholipid mixtures with a single gel phase, dipalmitoylphosphatidylcholine (DPPC)/cholesterol, gel/fluid phase coexistence DPPC/dioleoylphosphatidylcholine (DOPC), and ternary lipid mixtures containing cholesterol, mimicking lipid rafts (DOPC/DPPC/cholesterol and DOPC/sphingomyelin/cholesterol), i.e., with liquid ordered/liquid disordered (ld/lo) phase separation, were investigated. For all compositions studied, and in two different solid supports, mica and silicon, domain formation or rearrangement accompanied by lipid bilayer thinning and expansion was observed. In the case of gel/fluid coexistence, low ethanol concentrations lead to a marked thinning of the fluid but not of the gel domains. In the case of ld/lo all the bilayer thins simultaneously by a similar extent. In both cases, only the more disordered phase expanded significantly, indicating that ethanol increases the proportion of disordered domains. Water/bilayer interfacial tension variation and freezing point depression, inducing acyl chain disordering (including opening and looping), tilting, and interdigitation, are probably the main cause for the observed changes. The results presented herein demonstrate that ethanol influences the bilayer properties according to membrane lateral organization. Copyright © 2010 Elsevier B.V. All rights reserved.

Lipid Raft-Mediated Membrane Tethering and Delivery of Hydrophobic Cargos from Liquid Crystal-Based Nanocarriers.

PubMed

Nag, Okhil K; Naciri, Jawad; Oh, Eunkeu; Spillmann, Christopher M; Delehanty, James B

2016-04-20

A main goal of bionanotechnology and nanoparticle (NP)-mediated drug delivery (NMDD) continues to be the development of novel biomaterials that can controllably modulate the activity of the NP-associated therapeutic cargo. One of the desired subcellular locations for targeted delivery in NMDD is the plasma membrane. However, the controlled delivery of hydrophobic cargos to the membrane bilayer poses significant challenges including cargo precipitation and lack of specificity. Here, we employ a liquid crystal NP (LCNP)-based delivery system for the controlled partitioning of a model dye cargo from within the NP core into the plasma membrane bilayer. During synthesis of the NPs, the water-insoluble model dye cargo, 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO), was efficiently incorporated into the hydrophobic LCNP core as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) facilitated the localization of the dye-loaded NPs to lipid raft microdomains in the plasma membrane in HEK 293T/17 cell. Analysis of DiO cellular internalization kinetics revealed that when delivered as a LCNP-PEG-Chol NP, the half-life of DiO membrane residence time (30 min) was twice that of free DiO (DiO(free)) (15 min) delivered from bulk solution. Time-resolved laser scanning confocal microscopy was employed to visualize the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer as confirmed by Förster resonance energy transfer (FRET) imaging. Finally, the delivery of DiO as a LCNP-PEG-Chol complex resulted in the attenuation of its cytotoxicity; the NP form of DiO exhibited ∼30-40% less toxicity compared to DiO(free). Our data demonstrate the utility of the LCNP platform as an efficient vehicle for the combined membrane-targeted delivery and physicochemical modulation of molecular cargos using lipid raft-mediated tethering.

Prominin‐1/CD133: Lipid Raft Association, Detergent Resistance, and Immunodetection

PubMed Central

Karbanová, Jana; Lorico, Aurelio; Bornhäuser, Martin; Fargeas, Christine A.

2017-01-01

Summary The cell surface antigen prominin‐1 (alias CD133) has gained enormous interest in the past 2 decades and given rise to debates as to its utility as a biological stem and cancer stem cell marker. Important and yet often overlooked knowledge that is pertinent to its physiological function has been generated in other systems given its more general expression beyond primitive cells. This article briefly discusses the importance of particular biochemical features of CD133 with relation to its association with membrane microdomains (lipid rafts) and proper immunodetection. It also draws attention toward the adequate use of detergents and caveats that may apply to the interpretation of the results generated. Stem Cells Translational Medicine 2018;7:155–160 PMID:29271118

By activating Fas/ceramide synthase 6/p38 kinase in lipid rafts, Stichoposide D inhibits growth of leukemia xenografts

PubMed Central

Yun, Seong-Hoon; Park, Eun-Seon; Shin, Sung-Won; Ju, Mi-Ha; Han, Jin-Yeong; Jeong, Jin-Sook; Kim, Sung-Hyun; Stonik, Valentin A.; Kwak, Jong-Young; Park, Joo-In

2015-01-01

Stichoposide D (STD) is a marine triterpene glycoside isolated from sea cucumbers. We examined the molecular mechanisms underlying the antitumor activity of STD in human leukemia cells. The role of Fas (CD95), ceramide synthase 6 (CerS6) and p38 kinase during STD-induced apoptosis was examined in human leukemia cells. In addition, the antitumor effects of STD in K562 and HL-60 leukemia xenograft models were investigated. We found that STD induces Fas translocation to lipid rafts, and thus mediates cell apoptosis. We also observed the activation of CerS6 and p38 kinase during STD-induced apoptosis. The use of methyl-β-cyclodextrin and nystatin to disrupt lipid rafts prevents the clustering of Fas and the activation of CerS6 and p38 kinase, and also inhibits STD-induced apoptosis. Specific inhibition by Fas, CerS6, and p38 kinase siRNA transfection partially blocked STD-induced apoptosis. In addition, STD has antitumor activity through the activation of CerS6 and p38 kinase without displaying any toxicity in HL-60 and K562 xenograft models. We observed that the anti-tumor effect of STD is partially prevented in CerS6 shRNA-silenced xenograft models. We first report that Fas/CerS6/p38 kinase activation in lipid rafts by STD is involved in its anti-leukemic activity. We also established that STD is able to enhance the chemosensitivity of K562 cells to etoposide or Ara-C. These data suggest that STD may be used alone or in combination with other chemotherapeutic agents to treat leukemia. PMID:26318294

Time-Correlated Single-Photon Counting Fluorescence Imaging of Lipid Domains In Raft-Mimicking Giant Unilamellar Vesicles

NASA Astrophysics Data System (ADS)

Clarke, James; Cheng, Kwan; Shindell, Orrin; Wang, Exing

We have designed and constructed a high-throughput electrofusion chamber and an incubator to fabricate Giant Unilamellar Vesicles (GUVs) consisting of high-melting lipids, low-melting lipids, cholesterol and both ordered and disordered phase sensitive fluorescent probes (DiIC12, dehydroergosterol and BODIPY-Cholesterol). GUVs were formed in a 3 stage pulse sequence electrofusion process with voltages ranging from 50mVpp to 2.2Vpp and frequencies from 5Hz to 10Hz. Steady state and time-correlated single-photon counting (TCSPC) fluorescence lifetime (FLIM) based confocal and/or multi-photon microscopic techniques were used to characterize phase separated lipid domains in GUVs. Confocal imaging measures the probe concentration and the chemical environment of the system. TCSPC techniques determine the chemical environment through the perturbation of fluorescent lifetimes of the probes in the system. The above techniques will be applied to investigate the protein-lipid interactions involving domain formation. Specifically, the mechanisms governing lipid domain formations in the above systems that mimic the lipid rafts in cells will be explored. Murchison Fellowship at Trinity University.

Sphingosylphosphorylcholine promotes the differentiation of resident Sca-1 positive cardiac stem cells to cardiomyocytes through lipid raft/JNK/STAT3 and β-catenin signaling pathways.

PubMed

Li, Wenjing; Liu, Honghong; Liu, Pingping; Yin, Deling; Zhang, Shangli; Zhao, Jing

2016-07-01

Resident cardiac Sca-1-positive (+) stem cells may differentiate into cardiomyocytes to improve the function of damaged hearts. However, little is known about the inducers and molecular mechanisms underlying the myogenic conversion of Sca-1(+) stem cells. Here we report that sphingosylphosphorylcholine (SPC), a naturally occurring bioactive lipid, induces the myogenic conversion of Sca-1(+) stem cells, as evidenced by the increased expression of cardiac transcription factors (Nkx2.5 and GATA4), structural proteins (cardiac Troponin T), transcriptional enhancer (Mef2c) and GATA4 nucleus translocation. First, SPC activated JNK and STAT3, and the JNK inhibitor SP600125 or STAT3 inhibitor stattic impaired the SPC-induced expression of cardiac transcription factors and GATA4 nucleus translocation, which suggests that JNK and STAT3 participated in SPC-promoted cardiac differentiation. Moreover, STAT3 activation was inhibited by SP600125, whereas JNK was inhibited by β-cyclodextrin as a lipid raft breaker, which indicates a lipid raft/JNK/STAT3 pathway involved in SPC-induced myogenic transition. β-Catenin, degraded by activated GSK3β, was inhibited by SPC. Furthermore, GSK3β inhibitors weakened but the β-catenin inhibitor promoted SPC-induced differentiation. We found no crosstalk between the lipid raft/JNK/STAT3 and β-catenin pathway. Our study describes a lipid, SPC, as an endogenic inducer of myogenic conversion in Sca-1(+) stem cells with low toxicity and high efficiency for uptake. Copyright © 2016 Elsevier B.V. All rights reserved.

With or without rafts? Alternative views on cell membranes.

PubMed

Sevcsik, Eva; Schütz, Gerhard J

2016-02-01

The fundamental mechanisms of protein and lipid organization at the plasma membrane have continued to engage researchers for decades. Among proposed models, one idea has been particularly successful which assumes that sterol-dependent nanoscopic phases of different lipid chain order compartmentalize proteins, thereby modulating protein functionality. This model of membrane rafts has sustainably sparked the fields of membrane biophysics and biology, and shifted membrane lipids into the spotlight of research; by now, rafts have become an integral part of our terminology to describe a variety of cell biological processes. But is the evidence clear enough to continue supporting a theoretical concept which has resisted direct proof by observation for nearly twenty years? In this essay, we revisit findings that gave rise to and substantiated the raft hypothesis, discuss its impact on recent studies, and present alternative mechanisms to account for plasma membrane heterogeneity. © 2015 WILEY Periodicals, Inc.

Mutant RBL mast cells defective in Fc epsilon RI signaling and lipid raft biosynthesis are reconstituted by activated Rho-family GTPases.

PubMed

Field, K A; Apgar, J R; Hong-Geller, E; Siraganian, R P; Baird, B; Holowka, D

2000-10-01

Characterization of defects in a variant subline of RBL mast cells has revealed a biochemical event proximal to IgE receptor (Fc epsilon RI)-stimulated tyrosine phosphorylation that is required for multiple functional responses. This cell line, designated B6A4C1, is deficient in both Fc epsilon RI-mediated degranulation and biosynthesis of several lipid raft components. Agents that bypass receptor-mediated Ca(2+) influx stimulate strong degranulation responses in these variant cells. Cross-linking of IgE-Fc epsilon RI on these cells stimulates robust tyrosine phosphorylation but fails to mobilize a sustained Ca(2+) response. Fc epsilon RI-mediated inositol phosphate production is not detectable in these cells, and failure of adenosine receptors to mobilize Ca(2+) suggests a general deficiency in stimulated phospholipase C activity. Antigen stimulation of phospholipases A(2) and D is also defective. Infection of B6A4C1 cells with vaccinia virus constructs expressing constitutively active Rho family members Cdc42 and Rac restores antigen-stimulated degranulation, and active Cdc42 (but not active Rac) restores ganglioside and GPI expression. The results support the hypothesis that activation of Cdc42 and/or Rac is critical for Fc epsilon RI-mediated signaling that leads to Ca(2+) mobilization and degranulation. Furthermore, they suggest that Cdc42 plays an important role in the biosynthesis and expression of certain components of lipid rafts.

Carbon nanoparticles induce ceramide- and lipid raft-dependent signalling in lung epithelial cells: a target for a preventive strategy against environmentally-induced lung inflammation

PubMed Central

2012-01-01

Background Particulate air pollution in lung epithelial cells induces pathogenic endpoints like proliferation, apoptosis, and pro-inflammatory reactions. The activation of the epidermal growth factor receptor (EGFR) is a key event responsible for signalling events involving mitogen activated protein kinases specific for these endpoints. The molecular events leading to receptor activation however are not well understood. These events are relevant for the toxicological evaluation of inhalable particles as well as for potential preventive strategies in situations when particulate air pollution cannot be avoided. The current study therefore had the objective to elucidate membrane-coupled events leading to EGFR activation and the subsequent signalling cascade in lung epithelial cells. Furthermore, we aimed to identify the molecular target of ectoine, a biophysical active substance which we described to prevent carbon nanoparticle-induced lung inflammation. Methods Membrane signalling events were investigated in isolated lipid rafts from lung epithelial cells with regard to lipid and protein content of the signalling platforms. Using positive and negative intervention approaches, lipid raft changes, subsequent signalling events, and lung inflammation were investigated in vitro in lung epithelial cells (RLE-6TN) and in vivo in exposed animals. Results Carbon nanoparticle treatment specifically led to an accumulation of ceramides in lipid rafts. Detailed analyses demonstrated a causal link of ceramides and subsequent EGFR activation coupled with a loss of the receptor in the lipid raft fractions. In vitro and in vivo investigations demonstrate the relevance of these events for carbon nanoparticle-induced lung inflammation. Moreover, the compatible solute ectoine was able to prevent ceramide-mediated EGFR phosphorylation and subsequent signalling as well as lung inflammation in vivo. Conclusion The data identify a so far unknown event in pro-inflammatory signalling and

Raft-mediated trafficking of apical resident proteins occurs in both direct and transcytotic pathways in polarized hepatic cells: role of distinct lipid microdomains.

PubMed

Slimane, Tounsia Aït; Trugnan, Germain; Van IJzendoorn, Sven C D; Hoekstra, Dick

2003-02-01

In polarized hepatic cells, pathways and molecular principles mediating the flow of resident apical bile canalicular proteins have not yet been resolved. Herein, we have investigated apical trafficking of a glycosylphosphatidylinositol-linked and two single transmembrane domain proteins on the one hand, and two polytopic proteins on the other in polarized HepG2 cells. We demonstrate that the former arrive at the bile canalicular membrane via the indirect transcytotic pathway, whereas the polytopic proteins reach the apical membrane directly, after Golgi exit. Most importantly, cholesterol-based lipid microdomains ("rafts") are operating in either pathway, and protein sorting into such domains occurs in the biosynthetic pathway, largely in the Golgi. Interestingly, rafts involved in the direct pathway are Lubrol WX insoluble but Triton X-100 soluble, whereas rafts in the indirect pathway are both Lubrol WX and Triton X-100 insoluble. Moreover, whereas cholesterol depletion alters raft-detergent insolubility in the indirect pathway without affecting apical sorting, protein missorting occurs in the direct pathway without affecting raft insolubility. The data implicate cholesterol as a traffic direction-determining parameter in the direct apical pathway. Furthermore, raft-cargo likely distinguishing single vs. multispanning membrane anchors, rather than rafts per se (co)determine the sorting pathway.

Soluble Glucan Is Internalized and Trafficked to the Golgi Apparatus in Macrophages via a Clathrin-Mediated, Lipid Raft-Regulated Mechanism

PubMed Central

Goldman, Matthew P.; Kalbfleisch, John H.; Williams, David L.

2012-01-01

Glucans are natural product carbohydrates that stimulate immunity. Glucans are internalized by the pattern recognition receptor, Dectin-1. Glucans were thought to be trafficked to phagolysosomes, but this is unproven. We examined the internalization and trafficking of soluble glucans in macrophages. Incubation of macrophages with glucan resulted in internalization of Dectin-1 and glucan. Inhibition of clathrin blocked internalization of the Dectin-1/glucan complex. Lipid raft depletion resulted in decreased Dectin levels and glucan uptake. Once internalized, glucans colocalized with early endosomes at 0 to 15 min, with the Golgi apparatus at 15 min to 24 h, and with Dectin-1 immediately (0 h) and again later (15 min-24 h). Glucans did not colocalize with lysosomes at any time interval examined. We conclude that the internalization of Dectin-1/glucan complexes in macrophages is mediated by clathrin and negatively regulated by lipid rafts and/or caveolin-1. Upon internalization, soluble glucans are trafficked via endosomes to the Golgi apparatus, not lysosomes. PMID:22700434

Membrane segregation and downregulation of raft markers during sarcolemmal differentiation in skeletal muscle cells.

PubMed

Draeger, A; Monastyrskaya, K; Burkhard, F C; Wobus, A M; Moss, S E; Babiychuk, E B

2003-10-15

Muscle contraction implies flexibility in combination with force resistance and requires a high degree of sarcolemmal organization. Smooth muscle cells differentiate largely from mesenchymal precursor cells and gradually assume a highly periodic sarcolemmal organization. Skeletal muscle undergoes an even more striking differentiation programme, leading to cell fusion and alignment into myofibrils. The lipid bilayer of each cell type is further segregated into raft and non-raft microdomains of distinct lipid composition. Considering the extent of developmental rearrangement in skeletal muscle, we investigated sarcolemmal microdomain organization in skeletal and smooth muscle cells. The rafts in both muscle types are characterized by marker proteins belonging to the annexin family which localize to the inner membrane leaflet, as well as glycosyl-phosphatidyl-inositol (GPI)-anchored enzymes attached to the outer leaflet. We demonstrate that the profound structural rearrangements that occur during skeletal muscle maturation coincide with a striking decrease in membrane lipid segregation, downregulation of annexins 2 and 6, and a significant decrease in raft-associated 5'-nucleotidase activity. The relative paucity of lipid rafts in mature skeletal in contrast to smooth muscle suggests that the organization of sarcolemmal microdomains contributes to the muscle-specific differences in stimulatory responses and contractile properties.

Interfering With Lipid Raft Association: A Mechanism to Control Influenza Virus Infection By Sambucus Nigra

PubMed Central

Shahsavandi, Shahla; Ebrahimi, Mohammad Majid; Hasaninejad Farahani, Ameneh

2017-01-01

Sambucus nigra (elder) are broadly used species to treat microbial infections. The potential antiviral activity and mechanism action of elder fruit (EF) in human epithelium cell (A549) cultures infected with H9N2 influenza virus were determined. The effect of various concentrations of EF on influenza virus replication was examined by using virus titration, quantitative real time RT-PCR, fusion and lipid raft assays following two treatment procedures: A) pre-treated H9N2 virus with each concentration of EF extract and transfection of A549 cell cultures, and B) each concentrations of EF was added to H9N2 virus infected-cell cultures following virus adsorption. In both treatments with lower doses of EF increased viral titer as well as synthesized viral nucleoprotein as indicating the herb had no inhibitory effects on virus replication. In (B) trial with higher doses, 40 and 80 μg/mL of EF, a significant decrease in virus titer and viral protein synthesis were shown in EF treated cells indicating the herb affect either entry of viruses or inhibition virus particle release. The results suggest that EF treatment of the influenza virus infected-human epithelial cells may involve in lipid raft association which function as platform for formation of viral membrane fusion and budding. Differencesin treatment time and dose of EF extract in infected cells with influenza virus have a marked effect on the efficacy of the herb. PMID:29201101

Raft-mediated Trafficking of Apical Resident Proteins Occurs in Both Direct and Transcytotic Pathways in Polarized Hepatic Cells: Role of Distinct Lipid Microdomains

PubMed Central

Slimane, Tounsia Aït; Trugnan, Germain; van IJzendoorn, Sven C.D.; Hoekstra, Dick

2003-01-01

In polarized hepatic cells, pathways and molecular principles mediating the flow of resident apical bile canalicular proteins have not yet been resolved. Herein, we have investigated apical trafficking of a glycosylphosphatidylinositol-linked and two single transmembrane domain proteins on the one hand, and two polytopic proteins on the other in polarized HepG2 cells. We demonstrate that the former arrive at the bile canalicular membrane via the indirect transcytotic pathway, whereas the polytopic proteins reach the apical membrane directly, after Golgi exit. Most importantly, cholesterol-based lipid microdomains (“rafts”) are operating in either pathway, and protein sorting into such domains occurs in the biosynthetic pathway, largely in the Golgi. Interestingly, rafts involved in the direct pathway are Lubrol WX insoluble but Triton X-100 soluble, whereas rafts in the indirect pathway are both Lubrol WX and Triton X-100 insoluble. Moreover, whereas cholesterol depletion alters raft-detergent insolubility in the indirect pathway without affecting apical sorting, protein missorting occurs in the direct pathway without affecting raft insolubility. The data implicate cholesterol as a traffic direction-determining parameter in the direct apical pathway. Furthermore, raft-cargo likely distinguishing single vs. multispanning membrane anchors, rather than rafts per se (co)determine the sorting pathway. PMID:12589058

Complement component 1, q subcomponent binding protein (C1QBP) in lipid rafts mediates hepatic metastasis of pancreatic cancer by regulating IGF-1/IGF-1R signaling.

PubMed

Shi, Haojun; Fang, Winston; Liu, Minda; Fu, Deliang

2017-10-01

Pancreatic cancer shows a remarkable predilection for hepatic metastasis. Complement component 1, q subcomponent binding protein (C1QBP) can mediate growth factor-induced cancer cell chemotaxis and distant metastasis by activation of receptor tyrosine kinases. Coincidentally, insulin-like growth factor-1 (IGF-1) derived from the liver and cancer cells itself has been recognized as a critical inducer of hepatic metastasis. However, the mechanism underlying IGF-1-dependent hepatic metastasis of pancreatic cancer, in which C1QBP may be involved, remains unknown. In the study, we demonstrated a significant association between C1QBP expression and hepatic metastasis in patients with pancreatic cancer. IGF-1 induced the translocation of C1QBP from cytoplasm to lipid rafts and further drove the formation of CD44 variant 6 (CD44v6)/C1QBP complex in pancreatic cancer cells. C1QBP interacting with CD44v6 in lipid rafts promoted phosphorylation of IGF-1R and thus activated downstream PI3K and MAPK signaling pathways which mediated metastatic potential of pancreatic cancer cells including proliferation, apoptosis, invasion, adhesion and energy metabolism. Furthermore, C1QBP knockdown suppressed hepatic metastasis of pancreatic cancer cells in nude mice. We therefore conclude that C1QBP in lipid rafts serves a key regulator of IGF-1/IGF-1R-induced hepatic metastasis from pancreatic cancer. Our findings about C1QBP in lipid rafts provide a novel strategy to block IGF-1/IGF-1R signaling in pancreatic cancer and a reliable premise for more efficient combined modality therapies. © 2017 UICC.

Renal Na+-K+-Cl− cotransporter activity and vasopressin-induced trafficking are lipid raft-dependent

PubMed Central

Welker, Pia; Böhlick, Alexandra; Mutig, Kerim; Salanova, Michele; Kahl, Thomas; Schlüter, Hartmut; Blottner, Dieter; Ponce-Coria, Jose; Gamba, Gerardo; Bachmann, Sebastian

2008-01-01

Apical bumetanide-sensitive Na+-K+-2Cl− cotransporter (NKCC2), the kidney-specific member of a cation-chloride cotransporter superfamily, is an integral membrane protein responsible for the transepithelial reabsorption of NaCl. The role of NKCC2 is essential for renal volume regulation. Vasopressin (AVP) controls NKCC2 surface expression in cells of the thick ascending limb of the loop of Henle (TAL). We found that 40–70% of Triton X-100-insoluble NKCC2 was present in cholesterol-enriched lipid rafts (LR) in rat kidney and cultured TAL cells. The related Na+-Cl− cotransporter (NCC) from rat kidney was distributed in LR as well. NKCC2-containing LR were detected both intracellularly and in the plasma membrane. Bumetanide-sensitive transport of NKCC2 as analyzed by 86Rb+ influx in Xenopus laevis oocytes was markedly reduced by methyl-β-cyclodextrin (MβCD)-induced cholesterol depletion. In TAL, short-term AVP application induced apical vesicular trafficking along with a shift of NKCC2 from non-raft to LR fractions. In parallel, increased colocalization of NKCC2 with the LR ganglioside GM1 and their polar translocation were assessed by confocal analysis. Apical biotinylation showed twofold increases in NKCC2 surface expression. These effects were blunted by mevalonate-lovastatin/MβCD-induced cholesterol deprivation. Collectively, these findings demonstrate that a pool of NKCC2 distributes in rafts. Results are consistent with a model in which LR mediate polar insertion, activity, and AVP-induced trafficking of NKCC2 in the control of transepithelial NaCl transport. PMID:18579701

Study of Raft Domains in Model Membrane of DPPC/PE/Cholesterol

NASA Astrophysics Data System (ADS)

Lor, Chai; Hirst, Linda

2010-10-01

Raft domains in bilayer membrane are thought to play an important role in many cell functions such as cell signaling or trans-membrane protein activation. Here we use a model membrane consisting of DPPC/PE/cholesterol to examine the structure of membrane rafts and phase interactions. In particular we are interested in lipids containing the highly polyunsaturated fatty acid DHA. We use both atomic force microscopy (AFM) and fluorescence microscopy to obtain information on the structural properties of raft regions and track cholesterol. As expected, we find phase separation of raft regions between saturated and unsaturated lipids. Moreover, we find that the roughness of the domains change with varying cholesterol concentration possibly due to overpacking. This model study provides further understanding of the role of cholesterol in bilayer membrane leading towards a better knowledge of cell membranes.

Cholesterol Depletion Disorganizes Oocyte Membrane Rafts Altering Mouse Fertilization

PubMed Central

Buschiazzo, Jorgelina; Ialy-Radio, Come; Auer, Jana; Wolf, Jean-Philippe; Serres, Catherine

2013-01-01

Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-β-cyclodextrin. Cholesterol removal induced: (1) a decrease of the fertilization rate and index; and (2) a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol. PMID:23638166

PGE2 /EP4 Signaling Controls the Transfer of the Mammary Stem Cell State by Lipid Rafts in Extracellular Vesicles.

PubMed

Lin, Meng-Chieh; Chen, Shih-Yin; Tsai, Ho-Min; He, Pei-Lin; Lin, Yen-Chun; Herschman, Harvey; Li, Hua-Jung

2017-02-01

Prostaglandin E 2 (PGE 2 )-initiated signaling contributes to stem cell homeostasis and regeneration. However, it is unclear how PGE 2 signaling controls cell stemness. This study identifies a previously unknown mechanism by which PGE 2 /prostaglandin E receptor 4 (EP 4 ) signaling regulates multiple signaling pathways (e.g., PI3K/Akt signaling, TGFβ signaling, Wnt signaling, EGFR signaling) which maintain the basal mammary stem cell phenotype. A shift of basal mammary epithelial stem cells (MaSCs) from a mesenchymal/stem cell state to a non-basal-MaSC state occurs in response to prostaglandin E receptor 4 (EP 4 ) antagonism. EP 4 antagonists elicit release of signaling components, by controlling their trafficking into extracellular vesicles/exosomes in a lipid raft/caveolae-dependent manner. Consequently, EP 4 antagonism indirectly inactivates, through induced extracellular vesicle/exosome release, pathways required for mammary epithelial stem cell homeostasis, e.g. canonical/noncanonical Wnt, TGFβ and PI3K/Akt pathways. EP 4 antagonism causes signaling receptors and signaling components to shift from non-lipid raft fractions to lipid raft fractions, and to then be released in EP 4 antagonist-induced extracellular vesicles/exosomes, resulting in the loss of the stem cell state by mammary epithelial stem cells. In contrast, luminal mammary epithelial cells can acquire basal stem cell properties following ingestion of EP 4 antagonist-induced stem cell extracellular vesicles/exosomes, and can then form mammary glands. These findings demonstrate that PGE 2 /EP 4 signaling controls homeostasis of mammary epithelial stem cells through regulating extracellular vesicle/exosome release. Reprogramming of mammary epithelial cells can result from EP 4 -mediated stem cell property transfer by extracellular vesicles/exosomes containing caveolae-associated proteins, between mammary basal and luminal epithelial cells. Stem Cells 2017;35:425-444. © 2016 The Authors STEM CELLS

Raft membrane domains: from a liquid-ordered membrane phase to a site of pathogen attack.

PubMed

van der Goot, F G; Harder, T

2001-04-01

While the existence of cholesterol/sphingolipid (raft) membrane domains in the plasma membrane is now supported by strong experimental evidence, the structure of these domains, their size, their dynamics, and their molecular composition remain to be understood. Raft domains are thought to represent a specific physical state of lipid bilayers, the liquid-ordered phase. Recent observations suggest that in the mammalian plasma membrane small raft domains in ordered lipid phases are in a dynamic equilibrium with a less ordered membrane environment. Rafts may be enlarged and/or stabilized by protein-mediated cross-linking of raft-associated components. These changes of plasma membrane structure are perceived by the cells as signals, most likely an important element of immunoreceptor signalling. Pathogens abuse raft domains on the host cell plasma membrane as concentration devices, as signalling platforms and/or entry sites into the cell. Elucidation of these interactions requires a detailed understanding raft structure and dynamics. Copyright 2001 Academic Press.

Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane.

PubMed

Röper, K; Corbeil, D; Huttner, W B

2000-09-01

Membrane cholesterol-sphingolipid 'rafts', which are characterized by their insolubility in the non-ionic detergent Triton X-100 in the cold, have been implicated in the sorting of certain membrane proteins, such as placental alkaline phosphatase (PLAP), to the apical plasma membrane domain of epithelial cells. Here we show that prominin, an apically sorted pentaspan membrane protein, becomes associated in the trans-Golgi network with a lipid raft that is soluble in Triton X-100 but insoluble in another non-ionic detergent, Lubrol WX. At the cell surface, prominin remains insoluble in Lubrol WX and is selectively associated with microvilli, being largely segregated from the membrane subdomains containing PLAP. Cholesterol depletion results in the loss of prominin's microvillus-specific localization but does not lead to its complete intermixing with PLAP. We propose the coexistence within a membrane domain, such as the apical plasma membrane, of different cholesterol-based lipid rafts, which underlie the generation and maintenance of membrane subdomains.

Decreased levels of NMDA but not AMPA receptors in the lipid-raft fraction of 3xTg-AD model of Alzheimer's disease: Relation to Arc/Arg3.1 protein expression.

PubMed

Morin, Jean-Pascal; Díaz-Cintra, Sofía; Bermúdez-Rattoni, Federico; Delint-Ramírez, Ilse

2016-11-01

It was recently suggested that alteration in lipid raft composition in Alzheimer's disease may lead to perturbations in neurons signalosome, which may help explain the deficits observed in synaptic plasticity mechanisms and long-term memory impairments in AD models. As a first effort to address this issue, we evaluated lipid-raft contents of distinct NMDA and AMPA receptor subunits in the hippocampus of the 3xTg-AD model of Alzheimer's disease. Our results show that compared to controls, 10 months-old 3xTg-AD mice have diminished levels of NMDA receptors in rafts but not in post-synaptic density or total fractions. Additionally, the levels of GluR1 were unaltered in all the analyzed fractions. Finally, we went on to show that the diminished levels of NMDA receptors in rafts correlated with diminished global levels of Arc/Arg3.1, a synaptic protein with a central role in long-term memory formation. This study adds to our current understanding of the signaling pathways disruptions observed in current Alzheimer's disease models. Copyright © 2016 Elsevier Ltd. All rights reserved.

Detergent-Based Isolation of Yeast Membrane Rafts: An Inquiry-Based Laboratory Series for the Undergraduate Cell Biology or Biochemistry Lab

ERIC Educational Resources Information Center

Willhite, D. Grant; Wright, Stephen E.

2009-01-01

Lipid rafts have been implicated in numerous cellular processes including cell signaling, endocytosis, and even viral infection. Isolation of these lipid rafts often involves detergent treatment of the membrane to dissolve nonraft components followed by separation of raft regions in a density gradient. We present here an inquiry-based lab series…

Lipid raft components cholesterol and sphingomyelin increase H+/OH− permeability of phosphatidylcholine membranes

PubMed Central

Gensure, Rebekah H.; Zeidel, Mark L.; Hill, Warren G.

2006-01-01

H+/OH− permeation through lipid bilayers occurs at anomalously high rates and the determinants of proton flux through membranes are poorly understood. Since all life depends on proton gradients, it is important to develop a greater understanding of proton leak phenomena. We have used stopped-flow fluorimetry to probe the influence of two lipid raft components, chol (cholesterol) and SM (sphingomyelin), on H+/OH− and water permeability. Increasing the concentrations of both lipids in POPC (palmitoyl-2-oleoyl phosphatidylcholine) liposomes decreased water permeability in a concentration-dependent manner, an effect that correlated with increased lipid order. Surprisingly, proton flux was increased by increasing the concentration of chol and SM. The chol effect was complex with molar concentrations of 17.9, 33 and 45.7% giving 2.8-fold (P<0.01), 2.2-fold (P<0.001) and 5.1-fold (P<0.001) increases in H+/OH− permeability from a baseline of 2.4×10−2 cm/s. SM at 10 mole% effected a 2.8-fold increase (P<0.01), whereas 20 and 30 mole% enhanced permeability by 3.6-fold (P<0.05) and 4.1-fold respectively (P<0.05). Supplementing membranes containing chol with SM did not enhance H+/OH− permeability. Of interest was the finding that chol addition to soya-bean lipids decreased H+/OH− permeability, consistent with an earlier report [Ira and Krishnamoorthy (2001) J. Phys. Chem. B 105, 1484–1488]. We speculate that the presence of proton carriers in crude lipid extracts might contribute to this result. We conclude that (i) chol and SM specifically and independently increase rates of proton permeation in POPC bilayers, (ii) domains enriched in these lipids or domain interfaces may represent regions with high H+/OH− conductivity, (iii) H+/OH− fluxes are not governed by lipid order and (iv) chol can inhibit or promote H+/OH− permeability depending on the total lipid environment. Theories of proton permeation are discussed in the light of these results. PMID

Dynamin2, Clathrin, and Lipid Rafts Mediate Endocytosis of the Apical Na/K/2Cl Cotransporter NKCC2 in Thick Ascending Limbs*

PubMed Central

Ares, Gustavo R.; Ortiz, Pablo A.

2012-01-01

Steady-state surface levels of the apical Na/K/2Cl cotransporter NKCC2 regulate NaCl reabsorption by epithelial cells of the renal thick ascending limb (THAL). We reported that constitutive endocytosis of NKCC2 controls NaCl absorption in native THALs; however, the pathways involved in NKCC2 endocytosis are unknown. We hypothesized that NKCC2 endocytosis at the apical surface depends on dynamin-2 and clathrin. Measurements of steady-state surface NKCC2 and the rate of NKCC2 endocytosis in freshly isolated rat THALs showed that inhibition of endogenous dynamin-2 with dynasore blunted NKCC2 endocytosis by 56 ± 11% and increased steady-state surface NKCC2 by 67 ± 27% (p < 0.05). Expression of the dominant negative Dyn2K44A in THALs slowed the rate of NKCC2 endocytosis by 38 ± 8% and increased steady-state surface NKCC2 by 37 ± 8%, without changing total NKCC2 expression. Inhibition of clathrin-mediated endocytosis with chlorpromazine blunted NKCC2 endocytosis by 54 ± 6%, while preventing clathrin from interacting with synaptojanin also blunted NKCC2 endocytosis by 52 ± 5%. Disruption of lipid rafts blunted NKCC2 endocytosis by 39 ± 4% and silencing caveolin-1 by 29 ± 4%. Simultaneous inhibition of clathrin- and lipid raft-mediated endocytosis completely blocked NKCC2 internalization. We concluded that dynamin-2, clathrin, and lipid rafts mediate NKCC2 endocytosis and maintain steady-state apical surface NKCC2 in native THALs. These are the first data identifying the endocytic pathway for apical NKCC2 endocytosis. PMID:22977238

Impact of oxLDL on Cholesterol-Rich Membrane Rafts

PubMed Central

Levitan, Irena; Shentu, Tzu-Pin

2011-01-01

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. PMID:21490811

Inhibition of VEGF-dependent angiogenesis by the anti-CD82 monoclonal antibody 4F9 through regulation of lipid raft microdomains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nomura, Sayaka; Iwata, Satoshi; Hatano, Ryo

CD82 (also known as KAI1) belongs to the tetraspanin superfamily of type III transmembrane proteins, and is involved in regulating cell adhesion, migration and proliferation. In contrast to these well-established roles of CD82 in tumor biology, its function in endothelial cell (EC) activity and tumor angiogenesis is yet to be determined. In this study, we show that suppression of CD82 negatively regulates vascular endothelial growth factor (VEGF)-induced angiogenesis. Moreover, we demonstrate that the anti-CD82 mAb 4F9 effectively inhibits phosphorylation of VEGF receptor 2 (VEGFR2), which is the principal mediator of the VEGF-induced angiogenic signaling process in tumor angiogenesis, by regulatingmore » the organization of the lipid raft microdomain signaling platform in human EC. Our present work therefore suggests that CD82 on EC is a potential target for anti-angiogenic therapy in VEGFR2-dependent tumor angiogenesis. -- Highlights: •Knockdown of CD82 decreases EC migration, proliferation and angiogenesis. •Anti-CD82 mAb 4F9 inhibits EC migration, proliferation and angiogenesis. •4F9 inhibits VEGFR2 phosphorylation via control of CD82 distribution in lipid rafts.« less

High glucose upregulates BACE1-mediated Aβ production through ROS-dependent HIF-1α and LXRα/ABCA1-regulated lipid raft reorganization in SK-N-MC cells

PubMed Central

Lee, Hyun Jik; Ryu, Jung Min; Jung, Young Hyun; Lee, Sei-Jung; Kim, Jeong Yeon; Lee, Sang Hun; Hwang, In Koo; Seong, Je Kyung; Han, Ho Jae

2016-01-01

There is an accumulation of evidence indicating that the risk of Alzheimer’s disease is associated with diabetes mellitus, an indicator of high glucose concentrations in blood plasma. This study investigated the effect of high glucose on BACE1 expression and amyloidogenesis in vivo, and we present details of the mechanism associated with those effects. Our results, using ZLC and ZDF rat models, showed that ZDF rats have high levels of amyloid-beta (Aβ), phosphorylated tau, BACE1, and APP-C99. In vitro result with mouse hippocampal neuron and SK-N-MC, high glucose stimulated Aβ secretion and apoptosis in a dose-dependent manner. In addition, high glucose increased BACE1 and APP-C99 expressions, which were reversed by a reactive oxygen species (ROS) scavenger. Indeed, high glucose increased intracellular ROS levels and HIF-1α expression, associated with regulation of BACE1 and Liver X Receptor α (LXRα). In addition, high glucose induced ATP-binding cassette transporter A1 (ABCA1) down-regulation, was associated with LXR-induced lipid raft reorganization and BACE1 localization on the lipid raft. Furthermore, silencing of BACE1 expression was shown to regulate Aβ secretion and apoptosis of SK-N-MC. In conclusion, high glucose upregulates BACE1 expression and activity through HIF-1α and LXRα/ABCA1-regulated lipid raft reorganization, leading to Aβ production and apoptosis of SK-N-MC. PMID:27829662

Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Yijun; Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan; Pattnaik, Asit K.

2012-03-01

The porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 4 (GP4) resembles a typical type I membrane protein in its structure but lacks a hydrophilic tail at the C-terminus, suggesting that GP4 may be a lipid-anchored membrane protein. Using the human decay-accelerating factor (DAF; CD55), a known glycosyl-phosphatidylinositol (GPI) lipid-anchored protein, chimeric constructs were made to substitute the GPI-anchor domain of DAF with the putative lipid-anchor domain of GP4, and their membrane association and lipase cleavage were determined in cells. The DAF-GP4 fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC), indicating thatmore » the C-terminal domain of GP4 functions as a GPI anchor. Mutational studies for residues adjacent to the GPI modification site and characterization of respective mutant viruses generated from infectious cDNA clones show that the ability of GP4 for membrane association corresponded to virus viability and growth characteristics. The residues T158 ({omega} - 2, where {omega} is the GPI moiety at E160), P159 ({omega} - 1), and M162 ({omega} + 2) of GP4 were determined to be important for virus replication, with M162 being of particular importance for virus infectivity. The complete removal of the peptide-anchor domain in GP4 resulted in a complete loss of virus infectivity. The depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in PRRSV infection. Remarkably, GP4 was found to co-localize with CD163 in the lipid rafts on the plasma membrane. Since CD163 has been reported as a cellular receptor for PRRSV and GP4 has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus.« less

Fusion of raft-like lipid bilayers operated by a membranotropic domain of the HSV-type I glycoprotein gH occurs through a cholesterol-dependent mechanism.

PubMed

Vitiello, Giuseppe; Falanga, Annarita; Petruk, Ariel Alcides; Merlino, Antonello; Fragneto, Giovanna; Paduano, Luigi; Galdiero, Stefania; D'Errico, Gerardino

2015-04-21

A wealth of evidence indicates that lipid rafts are involved in the fusion of the viral lipid envelope with the target cell membrane. However, the interplay between these sterol- and sphingolipid-enriched ordered domains and viral fusion glycoproteins has not yet been clarified. In this work we investigate the molecular mechanism by which a membranotropic fragment of the glycoprotein gH of the Herpes Simplex Virus (HSV) type I (gH625) drives fusion of lipid bilayers formed by palmitoyl oleoyl phosphatidylcholine (POPC)-sphingomyelin (SM)-cholesterol (CHOL) (1 : 1 : 1 wt/wt/wt), focusing on the role played by each component. The comparative analysis of the liposome fusion assays, Dynamic Light Scattering (DLS), spectrofluorimetry, Neutron Reflectivity (NR) and Electron Spin Resonance (ESR) experiments, and Molecular Dynamics (MD) simulations shows that CHOL is fundamental for liposome fusion to occur. In detail, CHOL stabilizes the gH625-bilayer association by specific interactions with the peptide Trp residue. The interaction with gH625 causes an increased order of the lipid acyl chains, whose local rotational motion is significantly hampered. SM plays only a minor role in the process, favoring the propagation of lipid perturbation to the bilayer inner core. The stiffening of the peptide-interacting bilayer leaflet results in an asymmetric perturbation of the membrane, which is locally destabilized thus favoring fusion events. Our results show that viral fusion glycoproteins are optimally suited to exert a high fusogenic activity on lipid rafts and support the relevance of cholesterol as a key player of membrane-related processes.

Uptake of raft components into amyloid β-peptide aggregates and membrane damage.

PubMed

Sasahara, Kenji; Morigaki, Kenichi; Mori, Yasuko

2015-07-15

Amyloid aggregation and deposition of amyloid β-peptide (Aβ) are pathologic characteristics of Alzheimer's disease (AD). Recent reports have shown that the association of Aβ with membranes containing ganglioside GM1 (GM1) plays a pivotal role in amyloid deposition and the pathogenesis of AD. However, the molecular interactions responsible for membrane damage associated with Aβ deposition are not fully understood. In this study, we microscopically observed amyloid aggregation of Aβ in the presence of lipid vesicles and on a substrate-supported planar membrane containing raft components and GM1. The experimental system enabled us to observe lipid-associated aggregation of Aβ, uptake of the raft components into Aβ aggregates, and relevant membrane damage. The results indicate that uptake of raft components from the membrane into Aβ deposits induces macroscopic heterogeneity of the membrane structure. Copyright © 2015 Elsevier Inc. All rights reserved.

Lipid microdomains and the regulation of ion channel function

PubMed Central

Dart, Caroline

2010-01-01

Many types of ion channel localize to cholesterol and sphingolipid-enriched regions of the plasma membrane known as lipid microdomains or ‘rafts’. The precise physiological role of these unique lipid microenvironments remains elusive due largely to difficulties associated with studying these potentially extremely small and dynamic domains. Nevertheless, increasing evidence suggests that membrane rafts regulate channel function in a number of different ways. Raft-enriched lipids such as cholesterol and sphingolipids exert effects on channel activity either through direct protein–lipid interactions or by influencing the physical properties of the bilayer. Rafts also appear to selectively recruit interacting signalling molecules to generate subcellular compartments that may be important for efficient and selective signal transduction. Direct interaction with raft-associated scaffold proteins such as caveolin can also influence channel function by altering gating kinetics or by affecting trafficking and surface expression. Selective association of ion channels with specific lipid microenvironments within the membrane is thus likely to be an important and fundamental regulatory aspect of channel physiology. This brief review highlights some of the existing evidence for raft modulation of channel function. PMID:20519314

Effect of Dialkyl Ammonium Cationic Surfactants on the Microfluidity of Membranes Containing Raft Domains.

PubMed

Uyama, Makoto; Inoue, Kaori; Kinoshita, Koichi; Miyahara, Reiji; Yokoyama, Hirokazu; Nakano, Minoru

2018-01-01

It has been reported that a lot of receptors localize in lipid raft domains and that the microfluidity of these domains regulates the activation of these receptors. In this study, we focused on the lipid raft and in order to evaluate the physicochemical effects of surfactants on microfluidity of lipid membranes, we used liposomes comprising of egg-yolk L-α-phosphatidylcholine, egg-yolk sphingomyelin, and cholesterol as a model of cell membranes containing raft domains. The microfluidity of the domains was characterized by fluorescence spectrometry using 1,6-diphenyl-1,3,5-hexatriene and 2-dimethylamino-6-lauroylnaphthalene. Among several surfactants, dialkylammonium-type cationic surfactants most efficiently increased the microfluidity. It is therefore concluded that (1) the electrostatic interaction between the cationic surfactant and eggPC/eggSM/cholesterol liposome could be important, (2) surfactants with alkyl chains more effectively inserted into membranes than those with acyl chains, and (3) cationic surfactants with lower T m values have a greater ability to increase the fluidity.

Membrane microdomains, rafts, and detergent-resistant membranes in plants and fungi.

PubMed

Malinsky, Jan; Opekarová, Miroslava; Grossmann, Guido; Tanner, Widmar

2013-01-01

The existence of specialized microdomains in plasma membranes, postulated for almost 25 years, has been popularized by the concept of lipid or membrane rafts. The idea that detergent-resistant membranes are equivalent to lipid rafts, which was generally abandoned after a decade of vigorous data accumulation, contributed to intense discussions about the validity of the raft concept. The existence of membrane microdomains, meanwhile, has been verified by unequivocal independent evidence. This review summarizes the current state of research in plants and fungi with respect to common aspects of both kingdoms. In these organisms, principally immobile microdomains large enough for microscopic detection have been visualized. These microdomains are found in the context of cell-cell interactions (plant symbionts and pathogens), membrane transport, stress, and polarized growth, and the data corroborate at least three mechanisms of formation. As documented in this review, modern methods of visualization of lateral membrane compartments are also able to uncover the functional relevance of membrane microdomains.

Differential incorporation of docosahexaenoic acid into distinct cholesterol-rich membrane raft domains.

PubMed

Duraisamy, Yasotha; Lambert, Daniel; O'Neill, Catherine A; Padfield, Philip J

2007-09-07

We investigated the influence of docosahexaenoic acid (DHA) on the fatty acid and protein compositions of two populations of membrane rafts present in Caco-2 cells. DHA (100 microM) had no significant influence on the fatty acid or protein compositions of tight junction-associated, Lubrol insoluble, membrane rafts. However, DHA did significantly alter the fatty acid and protein compositions of "archetypal" Triton X-100 insoluble membrane rafts. The DHA content of the raft lipids increased 25-fold and was accompanied by a redistribution of src and fyn out of the rafts. DHA also increased Caco-2 cell monolayer permeability producing a 95% drop in transepithelial electrical resistance and a 8.56-fold increase in the flux of dextran. In conclusion, the data demonstrate that DHA does not increase permeability through modifying the TJ-associated rafts. The data do, however, show that DHA is differentially incorporated into different classes of membrane rafts, which has significant implications to our understanding of how omega-3 PUFAs modulate plasma membrane organization and cell function.

Prediction of glycolipid-binding domains from the amino acid sequence of lipid raft-associated proteins: application to HpaA, a protein involved in the adhesion of Helicobacter pylori to gastrointestinal cells.

PubMed

Fantini, Jacques; Garmy, Nicolas; Yahi, Nouara

2006-09-12

Protein-glycolipid interactions mediate the attachment of various pathogens to the host cell surface as well as the association of numerous cellular proteins with lipid rafts. Thus, it is of primary importance to identify the protein domains involved in glycolipid recognition. Using structure similarity searches, we could identify a common glycolipid-binding domain in the three-dimensional structure of several proteins known to interact with lipid rafts. Yet the three-dimensional structure of most raft-targeted proteins is still unknown. In the present study, we have identified a glycolipid-binding domain in the amino acid sequence of a bacterial adhesin (Helicobacter pylori adhesin A, HpaA). The prediction was based on the major properties of the glycolipid-binding domains previously characterized by structural searches. A short (15-mer) synthetic peptide corresponding to this putative glycolipid-binding domain was synthesized, and we studied its interaction with glycolipid monolayers at the air-water interface. The synthetic HpaA peptide recognized LacCer but not Gb3. This glycolipid specificity was in line with that of the whole bacterium. Molecular modeling studies gave some insights into this high selectivity of interaction. It also suggested that Phe147 in HpaA played a key role in LacCer recognition, through sugar-aromatic CH-pi stacking interactions with the hydrophobic side of the galactose ring of LacCer. Correspondingly, the replacement of Phe147 with Ala strongly affected LacCer recognition, whereas substitution with Trp did not. Our method could be used to identify glycolipid-binding domains in microbial and cellular proteins interacting with lipid shells, rafts, and other specialized membrane microdomains.

Interaction of chiral rafts in self-assembled colloidal membranes

NASA Astrophysics Data System (ADS)

Xie, Sheng; Hagan, Michael F.; Pelcovits, Robert A.

2016-03-01

Colloidal membranes are monolayer assemblies of rodlike particles that capture the long-wavelength properties of lipid bilayer membranes on the colloidal scale. Recent experiments on colloidal membranes formed by chiral rodlike viruses showed that introducing a second species of virus with different length and opposite chirality leads to the formation of rafts—micron-sized domains of one virus species floating in a background of the other viruses [Sharma et al., Nature (London) 513, 77 (2014), 10.1038/nature13694]. In this article we study the interaction of such rafts using liquid crystal elasticity theory. By numerically minimizing the director elastic free energy, we predict the tilt angle profile for both a single raft and two rafts in a background membrane, and the interaction between two rafts as a function of their separation. We find that the chiral penetration depth in the background membrane sets the scale for the range of the interaction. We compare our results with the experimental data and find good agreement for the strength and range of the interaction. Unlike the experiments, however, we do not observe a complete collapse of the data when rescaled by the tilt angle at the raft edge.

Fas/CD95 prevents autoimmunity independently of lipid raft localization and efficient apoptosis induction

PubMed Central

Cruz, Anthony C.; Ramaswamy, Madhu; Ouyang, Claudia; Klebanoff, Christopher A.; Sengupta, Prabuddha; Yamamoto, Tori N.; Meylan, Françoise; Thomas, Stacy K.; Richoz, Nathan; Eil, Robert; Price, Susan; Casellas, Rafael; Rao, V. Koneti; Lippincott-Schwartz, Jennifer; Restifo, Nicholas P.; Siegel, Richard M.

2016-01-01

Mutations affecting the apoptosis-inducing function of the Fas/CD95 TNF-family receptor result in autoimmune and lymphoproliferative disease. However, Fas can also costimulate T-cell activation and promote tumour cell growth and metastasis. Palmitoylation at a membrane proximal cysteine residue enables Fas to localize to lipid raft microdomains and induce apoptosis in cell lines. Here, we show that a palmitoylation-defective Fas C194V mutant is defective in inducing apoptosis in primary mouse T cells, B cells and dendritic cells, while retaining the ability to enhance naive T-cell differentiation. Despite inability to efficiently induce cell death, the Fas C194V receptor prevents the lymphoaccumulation and autoimmunity that develops in Fas-deficient mice. These findings indicate that induction of apoptosis through Fas is dependent on receptor palmitoylation in primary immune cells, and Fas may prevent autoimmunity by mechanisms other than inducing apoptosis. PMID:28008916

Coordinated Mechanosensitivity of Membrane Rafts and Focal Adhesions

PubMed Central

Fuentes, Daniela E.; Butler, Peter J.

2013-01-01

Endothelial cells sense mechanical forces of blood flow through mechanisms that involve focal adhesions (FAs). The mechanosensitive pathways that originate from FA-associated integrin activation may involve membrane rafts, small cholesterol- and sphigolipid-rich domains that are either immobilized, by virtue of their attachment to the cytoskeleton, or highly mobile in the plane of the plasma membrane. In this study, we fluorescently labeled non-mobile and mobile populations of GM1, a ganglioside associated with lipid rafts, and transfected cells with the red fluorescent protein-(RFP-) talin, an indicator of integrin activation at FAs, in order to determine the kinetics and sequential order of raft and talin mechanosensitivity. Cells were imaged under confocal microscopy during mechanical manipulation of a FA induced by a fibronectin (FN)-functionalized nanoelectrode with feedback control of position. First, FA deformation led to long range deformation of immobile rafts followed by active recoil of a subpopulation of displaced rafts. Second, initial adhesion between the FN-probe and the cell induced rapid accumulation of GM1 at the probe site with a time constant of 1.7 s. Talin accumulated approximately 20 s later with a time constant of 0.6 s. Third, a 1 μm deformation of the FA lead to immediate (0.3 s) increase in GM1 fluorescence and a later (6 s) increase in talin. Fourth, long term deformation of FAs led to continual GM1 accumulation at the probe site that was reversed upon removal of the deformation. These results demonstrate that rafts are directly mechanosensitive and that raft mobility may enable the earliest events related to FA mechanosensing and reinforcement upon force application. PMID:23487555

T1alpha/podoplanin shows raft-associated distribution in mouse lung alveolar epithelial E10 cells.

PubMed

Barth, Kathrin; Bläsche, Robert; Kasper, Michael

2010-01-01

T1alpha/(podoplanin) is abundantly expressed in the alveolar epithelial type I cells (ATI) of rodent and human lungs. Caveolin-1 is a classical primary structural protein of plasmalemal invaginations, so-called caveolae, which represent specialized lipid rafts, and which are particularly abundant in ATI cells. The biological functions of T1alpha in the alveolar epithelium are unknown. Here we report on the characteristics of raft domains in the microplicae/microvillar protrusions of ATI cells, which contain T1alpha. Detergent resistant membranes (DRMs) from cell lysates of the mouse epithelial ATI-like cell line E10 were prepared using different detergents followed by flotation in a sucrose gradient and tested by Western and dot blots with raft markers (caveolin-1, GM1) and nonraft markers (transferrin receptor, PDI and beta-Cop). Immunocytochemistry was employed for the localization of T1alpha in E10 cells and in situ in rat lungs. Our biochemical results showed that the solubility or insolubility of T1alpha and caveolin-1 differs in Triton X-100 and Lubrol WX, two distinct non-ionic detergents. Caveolin-1 was unsoluble in both detergents, whereas T1alpha was Triton X-100 soluble but Lubrol WX insoluble. Immunofluorescence double stainings revealed that both proteins were colocalized with GM1, while caveolin-1 and T1alpha were not colocalized in the plasma membrane. Cholesterol depletion modified the segregation of T1alpha in Lubrol WX DRMs. Cellular processes in ultrathin sections of cultured mouse E10 cells were immunogold positive. Immunoelectron microscopy (postembedding) of rat lung tissue revealed the preferential localization of T1alpha on apical microvillar protrusions of ATI cells. We conclude that T1alpha and caveolin-1 are located in distinct plasma membrane microdomains, which differ in their protein-lipid interactions. The raft-associated distribution of T1alpha may have an impact on a specific, not yet clarified function of this protein in the

Visualization of Detergent Solubilization of Membranes: Implications for the Isolation of Rafts

PubMed Central

Garner, Ashley E.; Smith, D. Alastair; Hooper, Nigel M.

2008-01-01

Although different detergents can give rise to detergent-resistant membranes of different composition, it is unclear whether this represents domain heterogeneity in the original membrane. We compared the mechanism of action of five detergents on supported lipid bilayers composed of equimolar sphingomyelin, cholesterol, and dioleoylphosphatidylcholine imaged by atomic force microscopy, and on raft and nonraft marker proteins in live cells imaged by confocal microscopy. There was a marked correlation between the detergent solubilization of the cell membrane and that of the supported lipid bilayers. In both systems Triton X-100 and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) distinguished between the nonraft liquid-disordered (ld) and raft liquid ordered (lo) lipid phases by selectively solubilizing the ld phase. A higher concentration of Lubrol was required, and not all the ld phase was solubilized. The solubilization by Brij 96 occurred by a two-stage mechanism that initially resulted in the solubilization of some ld phase and then progressed to the solubilization of both ld and lo phases simultaneously. Octyl glucoside simultaneously solubilized both lo and ld phases. These data show that the mechanism of membrane solubilization is unique to an individual detergent. Our observations have significant implications for using different detergents to isolate membrane rafts from biological systems. PMID:17933878

Membrane rafts stabilized by chiral liquid crystal correction to bare interfacial tension

NASA Astrophysics Data System (ADS)

Kang, Louis; Lubensky, T. C.

Lipid rafts are hypothesized to facilitate protein interaction, tension regulation, and trafficking in biological membranes, but the mechanisms responsible for their formation and maintenance are not clear. Recently, experiments showed that bidisperse mixtures of filamentous viruses can self-assemble into colloidal monolayers with thermodynamically stable rafts that exhibit chiral structure and repulsive interactions. We quantitatively explain these observations by modeling the membrane particles as chiral liquid crystals. Chiral twist promotes the formation of finite-sized rafts by decreasing the effective interfacial tension between rafts and background membrane. It also mediates a repulsion that distributes rafts evenly throughout the membrane. Although this system is composed of filamentous viruses whose aggregation is entropically driven by dextran depletants instead of phospholipids and cholesterol with prominent electrostatic interactions, colloidal and biological membranes share many of the same physical symmetries. Chiral twist can contribute to the behavior of both systems and may account for certain stereospecific effects observed in molecular membranes.

STIM1 converts TRPC1 from a receptor-operated to a store-operated channel: moving TRPC1 in and out of lipid rafts.

PubMed

Alicia, Sampieri; Angélica, Zepeda; Carlos, Saldaña; Alfonso, Salgado; Vaca, Luis

2008-11-01

While the role of members from the TRPC family of channels as receptor-operated channels (ROC) is well established and supported by numerous studies, the role of this family of channels as store-operated channels (SOC) has been the focus of a heated controversy over the last few years. In the present study, we have explored the modulation of STIM1 on human TRPC1 channel. We show that the association of STIM1 to TRPC1 favors the insertion of TRPC1 into lipid rafts, where TRPC1 functions as a SOC. In the absence of STIM1, TRPC1 associates to other members from the TRPC family of channels to form ROCs. A novel TIRFM-FRET method illustrates the relevance of the dynamic association between STIM1 and TRPC1 for the activation of SOC and the lipid raft localization of the STIM1-TRPC1 complex. This study provides new evidence about the dual activity of TRPC1 (forming ROC or SOC) and the partners needed to determine TRPC1 functional fate. It highlights also the role of plasma membrane microdomains and ER-PM junctions in modulating TRPC1 channel function and its association to STIM1.

Phagocytosis depends on TRPV2-mediated calcium influx and requires TRPV2 in lipids rafts: alteration in macrophages from patients with cystic fibrosis.

PubMed

Lévêque, Manuella; Penna, Aubin; Le Trionnaire, Sophie; Belleguic, Chantal; Desrues, Benoît; Brinchault, Graziella; Jouneau, Stéphane; Lagadic-Gossmann, Dominique; Martin-Chouly, Corinne

2018-03-09

Whereas many phagocytosis steps involve ionic fluxes, the underlying ion channels remain poorly defined. As reported in mice, the calcium conducting TRPV2 channel impacts the phagocytic process. Macrophage phagocytosis is critical for defense against pathogens. In cystic fibrosis (CF), macrophages have lost their capacity to act as suppressor cells and thus play a significant role in the initiating stages leading to chronic inflammation/infection. In a previous study, we demonstrated that impaired function of CF macrophages is due to a deficient phagocytosis. The aim of the present study was to investigate TRPV2 role in the phagocytosis capacity of healthy primary human macrophage by studying its activity, its membrane localization and its recruitment in lipid rafts. In primary human macrophages, we showed that P. aeruginosa recruits TRPV2 channels at the cell surface and induced a calcium influx required for bacterial phagocytosis. We presently demonstrate that to be functional and play a role in phagocytosis, TRPV2 might require a preferential localization in lipid rafts. Furthermore, CF macrophage displays a perturbed calcium homeostasis due to a defect in TRPV2. In this context, deregulated TRPV2-signaling in CF macrophages could explain their defective phagocytosis capacity that contribute to the maintenance of chronic infection.

Altered lipid raft–associated signaling and ganglioside expression in T lymphocytes from patients with systemic lupus erythematosus

PubMed Central

Jury, Elizabeth C.; Kabouridis, Panagiotis S.; Flores-Borja, Fabian; Mageed, Rizgar A.; Isenberg, David A.

2004-01-01

Systemic lupus erythematosus (SLE) is characterized by abnormalities in T lymphocyte receptor–mediated signal transduction pathways. Our previous studies have established that lymphocyte-specific protein tyrosine kinase (LCK) is reduced in T lymphocytes from patients with SLE and that this reduction is associated with disease activity and parallels an increase in LCK ubiquitination independent of T cell activation. This study investigated the expression of molecules that regulate LCK homeostasis, such as CD45, C-terminal Src kinase (CSK), and c-Cbl, in lipid raft domains from SLE T cells and investigated the localization of these proteins during T cell receptor (TCR) triggering. Our results indicate that the expression of raft-associated ganglioside, GM1, is increased in T cells from SLE patients and LCK may be differentially regulated due to an alteration in the association of CD45 with lipid raft domains. CD45 tyrosine phosphatase, which regulates LCK activity, was differentially expressed and its localization into lipid rafts was increased in T cells from patients with SLE. Furthermore, T cells allowed to “rest” in vitro showed a reversal of the changes in LCK, CD45, and GM1 expression. The results also revealed that alterations in the level of GM1 expression and lipid raft occupancy cannot be induced by serum factors from patients with SLE but indicated that cell-cell contact, activating aberrant proximal signaling pathways, may be important in influencing abnormalities in T cell signaling and, therefore, function in patients with SLE. PMID:15085197

Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin

PubMed Central

Schwarz, Karin; Simons, Matias; Reiser, Jochen; Saleem, Moin A.; Faul, Christian; Kriz, Wihelm; Shaw, Andrey S.; Holzman, Lawrence B.; Mundel, Peter

2001-01-01

NPHS2 was recently identified as a gene whose mutations cause autosomal recessive steroid-resistant nephrotic syndrome. Its product, podocin, is a new member of the stomatin family, which consists of hairpin-like integral membrane proteins with intracellular NH2- and COOH-termini. Podocin is expressed in glomerular podocytes, but its subcellular distribution and interaction with other proteins are unknown. Here we show, by immunoelectron microscopy, that podocin localizes to the podocyte foot process membrane, at the insertion site of the slit diaphragm. Podocin accumulates in an oligomeric form in lipid rafts of the slit diaphragm. Moreover, GST pull-down experiments reveal that podocin associates via its COOH-terminal domain with CD2AP, a cytoplasmic binding partner of nephrin, and with nephrin itself. That podocin interacts with CD2AP and nephrin in vivo is shown by coimmunoprecipitation of these proteins from glomerular extracts. Furthermore, in vitro studies reveal direct interaction of podocin and CD2AP. Hence, as with the erythrocyte lipid raft protein stomatin, podocin is present in high-order oligomers and may serve a scaffolding function. We postulate that podocin serves in the structural organization of the slit diaphragm and the regulation of its filtration function. PMID:11733557

Effects of cholesterol on plasma membrane lipid order in MCF-7 cells by two-photon microscopy

NASA Astrophysics Data System (ADS)

Zeng, Yixiu; Chen, Jianling; Yang, Hongqin; Wang, Yuhua; Li, Hui; Xie, Shusen

2014-09-01

Lipid rafts are cholesterol- and glycosphingolipids- enriched microdomains on plasma membrane surface of mammal cells, involved in a variety of cellular processes. Depleting cholesterol from the plasma membrane by drugs influences the trafficking of lipid raft markers. Optical imaging techniques are powerful tools to study lipid rafts in live cells due to its noninvasive feature. In this study, breast cancer cells MCF-7 were treated with different concentrations of MβCD to deplete cholesterol and an environmentally sensitive fluorescence probe, Laurdan was loaded to image lipid order by two-photon microscopy. The generalized polarization (GP) values were calculated to distinguish the lipid order and disorder phase. GP images and GP distributions of native and cholesterol-depleted MCF-7 cells were obtained. Our results suggest that even at low concentration (0.5 mM) of MβCD, the morphology of the MCF-7 cells changes. Small high GP areas (lipid order phase) decrease more rapidly than low GP areas (lipid disorder phase), indicating that lipid raft structure was altered more severely than nonraft domains. The data demonstrates that cholesterol dramatically affect raft coverage and plasma membrane fluidity in living cells.

Urokinase-type plasminogen activator receptor (uPAR) ligation induces a raft-localized integrin signaling switch that mediates the hypermotile phenotype of fibrotic fibroblasts.

PubMed

Grove, Lisa M; Southern, Brian D; Jin, Tong H; White, Kimberly E; Paruchuri, Sailaja; Harel, Efrat; Wei, Ying; Rahaman, Shaik O; Gladson, Candece L; Ding, Qiang; Craik, Charles S; Chapman, Harold A; Olman, Mitchell A

2014-05-02

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.

Mechanical Properties of Nanoscopic Lipid Domains

DOE PAGES

Nickels, Jonathan D.; Cheng, Xiaolin; Mostofian, Barmak; ...

2015-09-28

We found that the lipid raft hypothesis presents insight into how the cell membrane organizes proteins and lipids to accomplish its many vital functions. Yet basic questions remain about the physical mechanisms that lead to the formation, stability, and size of lipid rafts. Thus, much interest has been generated in the study of systems that contain similar lateral heterogeneities, or domains. In the current work we present an experimental approach that is capable of isolating the bending moduli of lipid domains. This is accomplished using neutron scattering and its unique sensitivity to the isotopes of hydrogen. Combining contrast matching approachesmore » with inelastic neutron scattering, we isolate the bending modulus of ~13 nm diameter domains residing in 60 nm unilamellar vesicles, whose lipid composition mimics the mammalian plasma membrane outer leaflet. Importantly, the bending modulus of the nanoscopic domains differs from the modulus of the continuous phase surrounding them. Moreover, from additional structural measurements and all-atom simulations, we also determine that nanoscopic domains are in-register across the bilayer leaflets. Taken together, these results inform a number of theoretical models of domain/raft formation and highlight the fact that mismatches in bending modulus must be accounted for when explaining the emergence of lateral heterogeneities in lipid systems and biological membranes.« less

Poly-lactic acid nanoparticles (PLA-NP) promote physiological modifications in lung epithelial cells and are internalized by clathrin-coated pits and lipid rafts.

PubMed

da Luz, Camila Macedo; Boyles, Matthew Samuel Powys; Falagan-Lotsch, Priscila; Pereira, Mariana Rodrigues; Tutumi, Henrique Rudolf; de Oliveira Santos, Eidy; Martins, Nathalia Balthazar; Himly, Martin; Sommer, Aniela; Foissner, Ilse; Duschl, Albert; Granjeiro, José Mauro; Leite, Paulo Emílio Corrêa

2017-01-31

Poly-lactic acid nanoparticles (PLA-NP) are a type of polymeric NP, frequently used as nanomedicines, which have advantages over metallic NP such as the ability to maintain therapeutic drug levels for sustained periods of time. Despite PLA-NP being considered biocompatible, data concerning alterations in cellular physiology are scarce. We conducted an extensive evaluation of PLA-NP biocompatibility in human lung epithelial A549 cells using high throughput screening and more complex methodologies. These included measurements of cytotoxicity, cell viability, immunomodulatory potential, and effects upon the cells' proteome. We used non- and green-fluorescent PLA-NP with 63 and 66 nm diameters, respectively. Cells were exposed with concentrations of 2, 20, 100 and 200 µg/mL, for 24, 48 and 72 h, in most experiments. Moreover, possible endocytic mechanisms of internalization of PLA-NP were investigated, such as those involving caveolae, lipid rafts, macropinocytosis and clathrin-coated pits. Cell viability and proliferation were not altered in response to PLA-NP. Multiplex analysis of secreted mediators revealed a low-level reduction of IL-12p70 and vascular epidermal growth factor (VEGF) in response to PLA-NP, while all other mediators assessed were unaffected. However, changes to the cells' proteome were observed in response to PLA-NP, and, additionally, the cellular stress marker miR155 was found to reduce. In dual exposures of staurosporine (STS) with PLA-NP, PLA-NP enhanced susceptibility to STS-induced cell death. Finally, PLA-NP were rapidly internalized in association with clathrin-coated pits, and, to a lesser extent, with lipid rafts. These data demonstrate that PLA-NP are internalized and, in general, tolerated by A549 cells, with no cytotoxicity and no secretion of pro-inflammatory mediators. However, PLA-NP exposure may induce modification of biological functions of A549 cells, which should be considered when designing drug delivery systems. Moreover

Functional Proteomic Analysis of Lipid Raft Kinase Complexes

DTIC Science & Technology

2009-08-01

Ribosomal protein SA Raft 12 46/300 + 16.0 8.0 2.0 18 8 2.3 14 6 2.3 712 + + IPI00023101 RQCD1 Homo sapiens protein involved in sexual development...KnownHuman 17-ODYA IPI00065486 ABCB6 CDNA FLJ32464 fis, clone SKNMC1000251, highly similar to Homo sapiens MT-ABCtransporter (MTABC) mRNA IPI00006675...3.7 : 0 + + IPI00023101 RQCD1 Homo sapiens protein involved in sexual development, complete cds + + IPI00021766 RTN4 Isoform 1 of Reticulon-4 12

Visualization of detergent solubilization of membranes: implications for the isolation of rafts.

PubMed

Garner, Ashley E; Smith, D Alastair; Hooper, Nigel M

2008-02-15

Although different detergents can give rise to detergent-resistant membranes of different composition, it is unclear whether this represents domain heterogeneity in the original membrane. We compared the mechanism of action of five detergents on supported lipid bilayers composed of equimolar sphingomyelin, cholesterol, and dioleoylphosphatidylcholine imaged by atomic force microscopy, and on raft and nonraft marker proteins in live cells imaged by confocal microscopy. There was a marked correlation between the detergent solubilization of the cell membrane and that of the supported lipid bilayers. In both systems Triton X-100 and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) distinguished between the nonraft liquid-disordered (l(d)) and raft liquid ordered (l(o)) lipid phases by selectively solubilizing the l(d) phase. A higher concentration of Lubrol was required, and not all the l(d) phase was solubilized. The solubilization by Brij 96 occurred by a two-stage mechanism that initially resulted in the solubilization of some l(d) phase and then progressed to the solubilization of both l(d) and l(o) phases simultaneously. Octyl glucoside simultaneously solubilized both l(o) and l(d) phases. These data show that the mechanism of membrane solubilization is unique to an individual detergent. Our observations have significant implications for using different detergents to isolate membrane rafts from biological systems.

Membrane-Associated Effects of Glucocorticoid on BACE1 Upregulation and Aβ Generation: Involvement of Lipid Raft-Mediated CREB Activation.

PubMed

Choi, Gee Euhn; Lee, Sei-Jung; Lee, Hyun Jik; Ko, So Hee; Chae, Chang Woo; Han, Ho Jae

2017-08-30

Glucocorticoid has been widely accepted to induce Alzheimer's disease, but the nongenomic effect of glucocorticoid on amyloid β (Aβ) generation has yet to be studied. Here, we investigated the effect of the nongenomic pathway induced by glucocorticoid on amyloid precursor protein processing enzymes as well as Aβ production using male ICR mice and human neuroblastoma SK-N-MC cells. Mice groups exposed to restraint stress or intracerebroventricular injection of Aβ showed impaired cognition, decreased intracellular glucocorticoid receptor (GR) level, but elevated level of membrane GR (mGR). In this respect, we identified the mGR-dependent pathway evoked by glucocorticoid using impermeable cortisol conjugated to BSA (cortisol-BSA) on SK-N-MC cells. Cortisol-BSA augmented the expression of β-site amyloid precursor protein cleaving enzyme 1 (BACE1), the level of C-terminal fragment β of amyloid precursor protein (C99) and Aβ production, which were maintained even after blocking intracellular GR. We also found that cortisol-BSA enhanced the interaction between mGR and Gαs, which colocalized in the lipid raft. The subsequently activated CREB by cortisol-BSA bound to the CRE site of the BACE1 promoter increasing its expression, which was downregulated by inhibiting CBP. Consistently, blocking CBP attenuated cognitive impairment and Aβ production induced by corticosterone treatment or intracerebroventricular injection of Aβ more efficiently than inhibiting intracellular GR in mice. In conclusion, glucocorticoid couples mGR with Gαs and triggers cAMP-PKA-CREB axis dependent on the lipid raft to stimulate BACE1 upregulation and Aβ generation. SIGNIFICANCE STATEMENT Patients with Alzheimer's disease (AD) have been growing sharply and stress is considered as the major environment factor of AD. Glucocorticoid is the primarily responsive factor to stress and is widely known to induce AD. However, most AD patients usually have impaired genomic pathway of glucocorticoid

Formation of supported lipid bilayers containing phase-segregated domains and their interaction with gold nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Melby, Eric S.; Mensch, Arielle C.; Lohse, Samuel E.

2016-01-01

The cell membrane represents an important biological interface that nanoparticles may encounter after being released into the environment. Interaction of nanoparticles with cellular membranes may alter membrane structure and function, lead to their uptake into cells, and elicit adverse biological responses. Supported lipid bilayers have proven to be valuable ex vivo models for biological membranes, allowing investigation of their mechanisms of interaction with nanoparticles with a degree of control impossible in living cells. To date, the majority of research on nanoparticle interaction with supported lipid bilayers has employed membranes composed of single or binary mixtures of phospholipids. Cellular membranes containmore » a wide variety of lipids and exhibit lateral organization. Ordered membrane domains enriched in specific membrane components are referred to as lipid rafts and have not been explored with respect to their interaction with nanoparticles. Here we develop model lipid raft-containing membranes amenable to investigation by a variety of surface-sensitive analytical techniques and demonstrate that lipid rafts influence the extent of nanoparticle attachment to model membranes. We determined conditions that allow reliable formation of bilayers containing rafts enriched in sphingomyelin and cholesterol and confirmed their morphology by structured illumination and atomic force microscopies. We demonstrate that lipid rafts increase attachment of cationic gold nanoparticles to model membranes under near physiological ionic strength conditions (0.1 M NaCl) at pH 7.4. We anticipate that these results will serve as the foundation for and motivate further study of nanoparticle interaction with compositionally varied lipid rafts.« less

DHA inhibited AGEs-induced retinal microglia activation via suppression of the PPARγ/NFκB pathway and reduction of signal transducers in the AGEs/RAGE axis recruitment into lipid rafts.

PubMed

Wang, Li; Chen, Ka; Liu, Kai; Zhou, Yong; Zhang, Ting; Wang, Bin; Mi, Mantian

2015-04-01

Recent studies revealed that dietary intake of docosahexaenoic acid (DHA) prevented diabetic retinopathy (DR), but the underlying mechanism was not fully understood. Retinal microglia are a specialized population of macrophages in retina. Considerable evidence has shown that microglia activation may trigger neuronal death and vascular dysfunction in DR. The aim of this study was to investigate the effects of DHA on advanced glycation end products (AGEs)-induced microglia activation using an in vitro microglia culture system, and concurrently to explore the mediating mechanisms. DHA inhibited AGEs-induced microglia activation and tumor necrosis factor α (TNFα) secretion. These effects of DHA were directly linked with suppression of nuclear factor-kappa B (NFκB) activity, as evident by the reduction of p-IκBα expression, p-NFκB p65 nucleus translocation, NFκB DNA binding activity, and the regulation of gene transcription (TNFα, IL-1β, ICAM-1, and RAGE mRNA). Furthermore, DHA significantly increased phosphorylation of peroxisome proliferator-activated receptor-gamma (PPARγ), and combined with PPARγ stealth RNAi oligonucleotide, we confirmed that DHA inhibition of AGEs-induced microglia activation was partially through the PPARγ/NFκB pathway. Moreover, although AGEs incubation dramatically elevated expression of the cell surface receptor for AGEs (RAGE), DHA significantly inhibited RAGE and Src recruitment into lipid rafts. The AGEs-RAGE axis downstream signal transducers increased mitogen-activated protein kinase (p38 and JNK) phosphorylation. Taken together, DHA might inhibit AGEs-induced microglia activation via suppression of the PPARγ/NFκB pathway, and reduction of RAGE and AGEs/RAGE transducer recruitment into lipid rafts. These results provide a novel potential mechanism for the anti-inflammatory effects of DHA in DR prevention.

Membrane raft organization is more sensitive to disruption by (n-3) PUFA than nonraft organization in EL4 and B cells.

PubMed

Rockett, Benjamin Drew; Franklin, Andrew; Harris, Mitchel; Teague, Heather; Rockett, Alexis; Shaikh, Saame Raza

2011-06-01

Model membrane and cellular detergent extraction studies show (n-3) PUFA predominately incorporate into nonrafts; thus, we hypothesized (n-3) PUFA could disrupt nonraft organization. The first objective of this study was to determine whether (n-3) PUFA disrupted nonrafts of EL4 cells, an extension of our previous work in which we discovered an (n-3) PUFA diminished raft clustering. EPA or DHA treatment of EL4 cells increased plasma membrane accumulation of the nonraft probe 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate by ~50-70% relative to a BSA control. Förster resonance energy transfer imaging showed EPA and DHA also disrupted EL4 nanometer scale nonraft organization by increasing the distance between nonraft molecules by ~25% compared with BSA. However, changes in nonrafts were due to an increase in cell size; under conditions where EPA or DHA did not increase cell size, nonraft organization was unaffected. We next translated findings on EL4 cells by testing if (n-3) PUFA administered to mice disrupted nonrafts and rafts. Imaging of B cells isolated from mice fed low- or high-fat (HF) (n-3) PUFA diets showed no change in nonraft organization compared with a control diet (CD). However, confocal microscopy revealed the HF (n-3) PUFA diet disrupted lipid raft clustering and size by ~40% relative to CD. Taken together, our data from 2 different model systems suggest (n-3) PUFA have limited effects on nonrafts. The ex vivo data, which confirm previous studies with EL4 cells, provide evidence that (n-3) PUFA consumed through the diet disrupt B cell lipid raft clustering.

The assembly of GM1 glycolipid- and cholesterol-enriched raft-like membrane microdomains is important for giardial encystation.

PubMed

De Chatterjee, Atasi; Mendez, Tavis L; Roychowdhury, Sukla; Das, Siddhartha

2015-05-01

Although encystation (or cyst formation) is an important step of the life cycle of Giardia, the cellular events that trigger encystation are poorly understood. Because membrane microdomains are involved in inducing growth and differentiation in many eukaryotes, we wondered if these raft-like domains are assembled by this parasite and participate in the encystation process. Since the GM1 ganglioside is a major constituent of mammalian lipid rafts (LRs) and known to react with cholera toxin B (CTXB), we used Alexa Fluor-conjugated CTXB and GM1 antibodies to detect giardial LRs. Raft-like structures in trophozoites are located in the plasma membranes and on the periphery of ventral discs. In cysts, however, they are localized in the membranes beneath the cyst wall. Nystatin and filipin III, two cholesterol-binding agents, and oseltamivir (Tamiflu), a viral neuraminidase inhibitor, disassembled the microdomains, as evidenced by reduced staining of trophozoites with CTXB and GM1 antibodies. GM1- and cholesterol-enriched LRs were isolated from Giardia by density gradient centrifugation and found to be sensitive to nystatin and oseltamivir. The involvement of LRs in encystation could be supported by the observation that raft inhibitors interrupted the biogenesis of encystation-specific vesicles and cyst production. Furthermore, culturing of trophozoites in dialyzed medium containing fetal bovine serum (which is low in cholesterol) reduced raft assembly and encystation, which could be rescued by adding cholesterol from the outside. Our results suggest that Giardia is able to form GM1- and cholesterol-enriched lipid rafts and these raft domains are important for encystation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Line tension at lipid phase boundaries as driving force for HIV fusion peptide-mediated fusion

NASA Astrophysics Data System (ADS)

Yang, Sung-Tae; Kiessling, Volker; Tamm, Lukas K.

2016-04-01

Lipids and proteins are organized in cellular membranes in clusters, often called `lipid rafts'. Although raft-constituent ordered lipid domains are thought to be energetically unfavourable for membrane fusion, rafts have long been implicated in many biological fusion processes. For the case of HIV gp41-mediated membrane fusion, this apparent contradiction can be resolved by recognizing that the interfaces between ordered and disordered lipid domains are the predominant sites of fusion. Here we show that line tension at lipid domain boundaries contributes significant energy to drive gp41-fusion peptide-mediated fusion. This energy, which depends on the hydrophobic mismatch between ordered and disordered lipid domains, may contribute tens of kBT to fusion, that is, it is comparable to the energy required to form a lipid stalk intermediate. Line-active compounds such as vitamin E lower line tension in inhomogeneous membranes, thereby inhibit membrane fusion, and thus may be useful natural viral entry inhibitors.

Membrane Raft Organization Is More Sensitive to Disruption by (n-3) PUFA Than Nonraft Organization in EL4 and B Cells123

PubMed Central

Rockett, Benjamin Drew; Franklin, Andrew; Harris, Mitchel; Teague, Heather; Rockett, Alexis; Shaikh, Saame Raza

2011-01-01

Model membrane and cellular detergent extraction studies show (n-3) PUFA predominately incorporate into nonrafts; thus, we hypothesized (n-3) PUFA could disrupt nonraft organization. The first objective of this study was to determine whether (n-3) PUFA disrupted nonrafts of EL4 cells, an extension of our previous work in which we discovered an (n-3) PUFA diminished raft clustering. EPA or DHA treatment of EL4 cells increased plasma membrane accumulation of the nonraft probe 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate by ~50–70% relative to a BSA control. Förster resonance energy transfer imaging showed EPA and DHA also disrupted EL4 nanometer scale nonraft organization by increasing the distance between nonraft molecules by ~25% compared with BSA. However, changes in nonrafts were due to an increase in cell size; under conditions where EPA or DHA did not increase cell size, nonraft organization was unaffected. We next translated findings on EL4 cells by testing if (n-3) PUFA administered to mice disrupted nonrafts and rafts. Imaging of B cells isolated from mice fed low- or high-fat (HF) (n-3) PUFA diets showed no change in nonraft organization compared with a control diet (CD). However, confocal microscopy revealed the HF (n-3) PUFA diet disrupted lipid raft clustering and size by ~40% relative to CD. Taken together, our data from 2 different model systems suggest (n-3) PUFA have limited effects on nonrafts. The ex vivo data, which confirm previous studies with EL4 cells, provide evidence that (n-3) PUFA consumed through the diet disrupt B cell lipid raft clustering. PMID:21525263

Citrulline diet supplementation improves specific age-related raft changes in wild-type rodent hippocampus.

PubMed

Marquet-de Rougé, Perrine; Clamagirand, Christine; Facchinetti, Patricia; Rose, Christiane; Sargueil, Françoise; Guihenneuc-Jouyaux, Chantal; Cynober, Luc; Moinard, Christophe; Allinquant, Bernadette

2013-10-01

The levels of molecules crucial for signal transduction processing change in the brain with aging. Lipid rafts are membrane microdomains involved in cell signaling. We describe here substantial biophysical and biochemical changes occurring within the rafts in hippocampus neurons from aging wild-type rats and mice. Using continuous sucrose density gradients, we observed light-, medium-, and heavy raft subpopulations in young adult rodent hippocampus neurons containing very low levels of amyloid precursor protein (APP) and almost no caveolin-1 (CAV-1). By contrast, old rodents had a homogeneous age-specific high-density caveolar raft subpopulation containing significantly more cholesterol (CHOL), CAV-1, and APP. C99-APP-Cter fragment detection demonstrates that the first step of amyloidogenic APP processing takes place in this caveolar structure during physiological aging of the rat brain. In this age-specific caveolar raft subpopulation, levels of the C99-APP-Cter fragment are exponentially correlated with those of APP, suggesting that high APP concentrations may be associated with a risk of large increases in beta-amyloid peptide levels. Citrulline (an intermediate amino acid of the urea cycle) supplementation in the diet of aged rats for 3 months reduced these age-related hippocampus raft changes, resulting in raft patterns tightly close to those in young animals: CHOL, CAV-1, and APP concentrations were significantly lower and the C99-APP-Cter fragment was less abundant in the heavy raft subpopulation than in controls. Thus, we report substantial changes in raft structures during the aging of rodent hippocampus and describe new and promising areas of investigation concerning the possible protective effect of citrulline on brain function during aging.

Life raft stabilizer

NASA Technical Reports Server (NTRS)

Radnofsky, M. I.; Barnett, J. H., Jr.; Harrison, F. L.; Marak, R. J. (Inventor)

1973-01-01

An improved life raft stabilizer for reducing rocking and substantially precluding capsizing is discussed. The stabilizer may be removably attached to the raft and is defined by flexible side walls which extend a considerable depth downwardly to one another in the water. The side walls, in conjunction with the floor of the raft, form a ballast enclosure. A weight is placed in the bottom of the enclosure and water port means are provided in the walls. Placement of the stabilizer in the water allows the weighted bottom to sink, producing submerged deployment thereof and permitting water to enter the enclosure through the port means, thus forming a ballast for the raft.

Lipid Rafts Assemble Dynein Ensembles.

PubMed

Nirschl, Jeffrey J; Ghiretti, Amy E; Holzbaur, Erika L F

2016-05-01

New work by Rai et al. identifies a novel mechanism regulating phagosome transport in cells: the clustering of dynein motors into lipid microdomains, leading to enhanced unidirectional motility. Clustering may be especially important for dynein, a motor that works most efficiently in teams. Copyright © 2016 Elsevier Ltd. All rights reserved.

Apo AI/ABCA1-dependent and HDL3-mediated lipid efflux from compositionally distinct cholesterol-based microdomains.

PubMed

Drobnik, Wolfgang; Borsukova, Hana; Böttcher, Alfred; Pfeiffer, Alexandra; Liebisch, Gerhard; Schütz, Gerhard J; Schindler, Hansgeorg; Schmitz, Gerd

2002-04-01

We have investigated whether a raft heterogeneity exists in human monocyte-derived macrophages and fibroblasts and whether these microdomains are modulated by lipid efflux. Triton X-100 (Triton) or Lubrol WX (Lubrol) detergent-resistant membranes from cholesterol-loaded monocytes were associated with the following findings: (i) Lubrol-DRM contained most of the cellular cholesterol and at least 75% of Triton-detergent-resistant membranes. (ii) 'Lubrol rafts', defined by their solubility in Triton but insolubility in Lubrol, were enriched in unsaturated phosphatidylcholine and showed a lower cholesterol to choline-phospholipid ratio compared to Triton rafts. (iii) CD14 and CD55 were recovered in Triton- and Lubrol-detergent-resistant membranes, whereas CD11b was found exclusively in Triton DRM. ABCA1 implicated in apo AI-mediated lipid efflux and CDC42 were partially localized in Lubrol- but not in Triton-detergent-resistant membranes. (iv) Apo AI preferentially depleted cholesterol and choline-phospholipids from Lubrol rafts, whereas HDL3 additionally decreased the cholesterol content of Triton rafts. In fibroblasts, neither ABCA1 nor CDC42 was found in Lubrol rafts, and both apo AI and HDL3 reduced the lipid content in Lubrol- as well as in Triton-detergent-resistant membranes. In summary, we provide evidence for the existence of compositionally distinct membrane microdomains in human cells and their modulation by apo AI/ABCA1-dependent and HDL3-mediated lipid efflux.

A comparative study on the raft chemical properties of various alginate antacid raft-forming products.

PubMed

Dettmar, Peter W; Gil-Gonzalez, Diana; Fisher, Jeanine; Flint, Lucy; Rainforth, Daniel; Moreno-Herrera, Antonio; Potts, Mark

2018-01-01

Research to measure the chemical characterization of alginate rafts for good raft performance and ascertain how formulation can affect chemical parameters. A selection of alginate formulations was investigated all claiming to be proficient raft formers with significance between products established and ranked. Procedures were selected which demonstrated the chemical characterization allowing rafts to effectively impede the reflux into the esophagus or in severe cases to be refluxed preferentially into the esophagus and exert a demulcent effect, with focus of current research on methods which complement previous studies centered on physical properties. The alginate content was analyzed by a newly developed HPLC method. Methods were used to determine the neutralization profile and the acid neutralization within the raft determined along with how raft structure affects neutralization. Alginate content of Gaviscon Double Action (GDA) within the raft was significantly superior (p < .0001) to all competitor products. The two products with the highest raft acid neutralization capacity were GDA and Rennie Duo, the latter product not being a raft former. Raft structure was key and GDA had the right level of porosity to allow for longer duration of neutralization. Alginate formulations require three chemical reactions to take place simultaneously: transformation to alginic acid, sodium carbonate reacting to form carbon dioxide, calcium releasing free calcium ions to bind with alginic acid providing strength to raft formation. GDA was significantly superior (p <.0001) to all other comparators.

Skeleton-Controlled pDNA Delivery of Renewable Steroid-Based Cationic Lipids, the Endocytosis Pathway Analysis and Intracellular Localization

PubMed Central

Wang, Zhao; Luo, Ting; Cao, Amin; Sun, Jingjing

2018-01-01

Using renewable and biocompatible natural-based resources to construct functional biomaterials has attracted great attention in recent years. In this work, we successfully prepared a series of steroid-based cationic lipids by integrating various steroid skeletons/hydrophobes with (l-)-arginine headgroups via facile and efficient synthetic approach. The plasmid DNA (pDNA) binding affinity of the steroid-based cationic lipids, average particle sizes, surface potentials, morphologies and stability of the steroid-based cationic lipids/pDNA lipoplexes were disclosed to depend largely on the steroid skeletons. Cellular evaluation results revealed that cytotoxicity and gene transfection efficiency of the steroid-based cationic lipids in H1299 and HeLa cells strongly relied on the steroid hydrophobes. Interestingly, the steroid lipids/pDNA lipoplexes inclined to enter H1299 cells mainly through caveolae and lipid-raft mediated endocytosis pathways, and an intracellular trafficking route of “lipid-raft-mediated endocytosis→lysosome→cell nucleic localization” was accordingly proposed. The study provided possible approach for developing high-performance steroid-based lipid gene carriers, in which the cytotoxicity, gene transfection capability, endocytosis pathways, and intracellular trafficking/localization manners could be tuned/controlled by introducing proper steroid skeletons/hydrophobes. Noteworthy, among the lipids, Cho-Arg showed remarkably high gene transfection efficacy, even under high serum concentration (50% fetal bovine serum), making it an efficient gene transfection agent for practical application. PMID:29373505

Sizes of lipid domains: What do we know from artificial lipid membranes? What are the possible shared features with membrane rafts in cells?

PubMed

Rosetti, Carla M; Mangiarotti, Agustín; Wilke, Natalia

2017-05-01

In model lipid membranes with phase coexistence, domain sizes distribute in a very wide range, from the nanometer (reported in vesicles and supported films) to the micrometer (observed in many model membranes). Domain growth by coalescence and Ostwald ripening is slow (minutes to hours), the domain size being correlated with the size of the capture region. Domain sizes thus strongly depend on the number of domains which, in the case of a nucleation process, depends on the oversaturation of the system, on line tension and on the perturbation rate in relation to the membrane dynamics. Here, an overview is given of the factors that affect nucleation or spinodal decomposition and domain growth, and their influence on the distribution of domain sizes in different model membranes is discussed. The parameters analyzed respond to very general physical rules, and we therefore propose a similar behavior for the rafts in the plasma membrane of cells, but with obstructed mobility and with a continuously changing environment. Copyright © 2017 Elsevier B.V. All rights reserved.

Controlling Styrene Maleic Acid Lipid Particles through RAFT.

PubMed

Smith, Anton A A; Autzen, Henriette E; Laursen, Tomas; Wu, Vincent; Yen, Max; Hall, Aaron; Hansen, Scott D; Cheng, Yifan; Xu, Ting

2017-11-13

The ability of styrene maleic acid copolymers to dissolve lipid membranes into nanosized lipid particles is a facile method of obtaining membrane proteins in solubilized lipid discs while conserving part of their native lipid environment. While the currently used copolymers can readily extract membrane proteins in native nanodiscs, their highly disperse composition is likely to influence the dispersity of the discs as well as the extraction efficiency. In this study, reversible addition-fragmentation chain transfer was used to control the polymer architecture and dispersity of molecular weights with a high-precision. Based on Monte Carlo simulations of the polymerizations, the monomer composition was predicted and allowed a structure-function analysis of the polymer architecture, in relation to their ability to assemble into lipid nanoparticles. We show that a higher degree of control of the polymer architecture generates more homogeneous samples. We hypothesize that low dispersity copolymers, with control of polymer architecture are an ideal framework for the rational design of polymers for customized isolation and characterization of integral membrane proteins in native lipid bilayer systems.

Lipid raft-like liposomes used for targeted delivery of a chimeric entry-inhibitor peptide with anti-HIV-1 activity.

PubMed

Gómara, María José; Pérez-Pomeda, Ignacio; Gatell, José María; Sánchez-Merino, Victor; Yuste, Eloisa; Haro, Isabel

2017-02-01

The work reports the design and synthesis of a chimeric peptide that is composed of the peptide sequences of two entry inhibitors which target different sites of HIV-1 gp41. The chimeric peptide offers the advantage of targeting two gp41 regions simultaneously: the fusion peptide and the loop both of which are membrane active and participate in the membrane fusion process. We therefore use lipid raft-like liposomes as a tool to specifically direct the chimeric inhibitor peptide to the membrane domains where the HIV-1 envelope protein is located. Moreover, the liposomes that mimic the viral membrane composition protect the chimeric peptide against proteolytic digestion thereby increasing the stability of the peptide. The described liposome preparations are suitable nanosystems for managing hydrophobic entry-inhibitor peptides as putative therapeutics. Copyright © 2016 Elsevier Inc. All rights reserved.

Self Righting Life Raft

NASA Technical Reports Server (NTRS)

1982-01-01

The Givens Buoy Raft was designed and manufactured for inventor Jim Givens of Givens Marine Survival Co. Inc., by RPR Industries, Inc. The Raft consists of a canopied topside and an underwater hemispheric ballast chamber. It has a heavy ballast stabilization system, adopted from NASA technology, which negates the capsizing problem. A "flapper valve" admits large amounts of water to the hemisphere chamber providing ballast to keep the center of gravity constant; stabilization system compensates for changes in wave angle and weight shifting of raft occupants. Mr. Givens has an exclusive patent license for use of the NASA technology. Produced in various sizes, capacities range from six to 20 persons. Raft is housed in a canister, available in several configurations. A pull on a line triggers the automatic inflation process, which takes 12 seconds. The raft has been credited with saving 230 lives in the last five years. It has found wide acceptance with operators of fishing boats, pleasure craft and other vessels. The Coast Guard is purchasing the rafts for use on its rescue helicopters and the Navy has a development program to adapt the system. The Coast Guard last year announced a proposed amendment of its regulations that would require large ballast chambers on inflatable life rafts.

The insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) alters the membrane raft location of the TSH receptor stably expressed in Chinese hamster ovary cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Gregorio, Francesca; Pellegrino, Mario; Picchietti, Simona

2011-06-01

DDT is a highly lipophilic molecule known to deplete membrane rafts of their phosphoglycolipid and cholesterol contents. However, we have recently shown that DDT can also alter the thyroid homeostasis by inhibiting TSH receptor (TSHr) internalization. The present study was undertaken to verify whether DDT goitrogenic effects are due to the insecticide acting directly on TSHr or via alteration of the membrane rafts hosting the receptor itself. Our results demonstrate that, in CHO-TSHr transfected cells, TSHr is activated in the presence of TSH, while it is inhibited following DDT exposure. DDT can also reduce the endocytic vesicular traffic, alter themore » extension of multi-branched microvilli along their plasma membranes and induce TSHr shedding in vesicular forms. To verify whether TSHr displacement might depend on DDT altering the raft constitution of CHO-TSHr cell membranes the extent of TSHr and lipid raft co-localization was examined by confocal microscopy. Evidence shows that receptor/raft co-localization increased significantly upon exposure to TSH, while receptors and lipid rafts become dislodged on opposite cell poles in DDT-exposed CHO-TSHr cells. As a control, under similar culturing conditions, diphenylethylene, which is known to be a lipophilic substance that is structurally related to DDT, did not affect the extent of TSHr and lipid raft co-localization in CHO-TSHr cells treated with TSH. These findings corroborate and extend our view that, in CHO cells, the DDT disrupting action on TSHr is primarily due to the insecticide acting on membranes to deplete their raft cholesterol content, and that the resulting inhibition on TSHr internalization is due to receptor dislodgement from altered raft microdomains of the plasma membrane. - Highlights: >DDT is a pesticide with a severe environmental impact >Epidemiologic correlation exists between exposition to DDT and thyroid dysfunction >DDT is a lipophilic molecule that has been shown to inhibit TSH

A novel trigger for cholesterol-dependent smooth muscle contraction mediated by the sphingosylphosphorylcholine-Rho-kinase pathway in the rat basilar artery: a mechanistic role for lipid rafts.

PubMed

Shirao, Satoshi; Yoneda, Hiroshi; Shinoyama, Mizuya; Sugimoto, Kazutaka; Koizumi, Hiroyasu; Ishihara, Hideyuki; Oka, Fumiaki; Sadahiro, Hirokazu; Nomura, Sadahiro; Fujii, Masami; Tamechika, Masakatsu; Kagawa, Yoshiteru; Owada, Yuji; Suzuki, Michiyasu

2015-05-01

Hyperlipidemia is a risk factor for abnormal cerebrovascular events. Rafts are cholesterol-enriched membrane microdomains that influence signal transduction. We previously showed that Rho-kinase-mediated Ca(2+) sensitization of vascular smooth muscle (VSM) induced by sphingosylphosphorylcholine (SPC) has a pivotal role in cerebral vasospasm. The goals of the study were to show SPC-Rho-kinase-mediated VSM contraction in vivo and to link this effect to cholesterol and rafts. The SPC-induced VSM contraction measured using a cranial window model was reversed by Y-27632, a Rho-kinase inhibitor, in rats fed a control diet. The extent of SPC-induced contraction correlated with serum total cholesterol. Total cholesterol levels in the internal carotid artery (ICA) were significantly higher in rats fed a cholesterol diet compared with a control diet or a β-cyclodextrin diet, which depletes VSM cholesterol. Western blotting and real-time PCR revealed increases in flotillin-1, a raft marker, and flotillin-1 mRNA in the ICA in rats fed a cholesterol diet, but not in rats fed the β-cyclodextrin diet. Depletion of cholesterol decreased rafts in VSM cells, and prevention of an increase in cholesterol by β-cyclodextrin inhibited SPC-induced contraction in a cranial window model. These results indicate that cholesterol potentiates SPC-Rho-kinase-mediated contractions of importance in cerebral vasospasm and are compatible with a role for rafts in this process.

Lubrol-RAFTs in melanoma cells: a molecular platform for tumor-promoting ephrin-B2-integrin-beta1 interaction.

PubMed

Meyer, Stefanie; Orsó, Evelyn; Schmitz, Gerd; Landthaler, Michael; Vogt, Thomas

2007-07-01

Ephrins control cell motility and matrix adhesion. These functions play a pivotal role in cancer progression, for example, in malignant melanomas. We have previously shown that the ephrin-B2-tumor-promoting action is partly mediated by integrin-beta1 interaction. However, the subcellular prerequisites for molecular interaction like molecular proximity and co-compartmentalization have not been elucidated yet. Specific cholesterol-rich microdomains, termed lipid rafts (RAFTs), are known to be essential for functional ephrin-B2 signalling and integrin-mediated effects. Therefore, we addressed the question whether RAFT co-compartmentalization of both molecules could provide the molecular platform for their tumor-promoting interaction. In this study, we show that overexpressed ephrin-B2 is not only compartmentalized to classical Triton X-100 RAFTs in B16 melanoma cells, but also to the recently defined Lubrol-RAFTs. Interestingly, in the melanoma cells investigated, integrin-beta1 is also preferentially detected in such Lubrol-RAFTs. Accordingly, the presence of ephrin-B2 and integrin-beta1 in RAFTs and their function in cell migration and matrix attachment are highly sensitive to RAFT disruption by cholesterol depletion. Confocal fluorescence microscopy analyses also support the concept of a close molecular proximity and functional interplay of ephrin-B2 and integrin-beta1 in the plasma membrane. We conclude that Lubrol-RAFTs probably represent the platform for tumor-promoting ephrin-B2-integrin-beta1 interaction, which could become an interesting target for future antitumoral therapies.

Association of membrane/lipid rafts with the platelet cytoskeleton and the caveolin PY14: participation in the adhesion process.

PubMed

Cerecedo, Doris; Martínez-Vieyra, Ivette; Maldonado-García, Deneb; Hernández-González, Enrique; Winder, Steve J

2015-11-01

Platelets are the most prominent elements of blood tissue involved in hemostasis at sites of blood vessel injury. Platelet cytoskeleton is responsible for their shape modifications observed during activation and adhesion to the substratum; therefore the interactions between cytoskeleton and plasma membrane are critical to modulate blood platelet functions. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to membrane/lipid rafts (MLR) and regulate lateral diffusion of membrane proteins and lipids. Resting, thrombin-activated, and adherent human platelets were processed for biochemical studies including western-blot and immunprecipitation assays and confocal analysis were performed to characterize the interaction of MLR with the main cytoskeleton elements and β-dystroglycan as well as with the association of caveolin-1 PY14 with focal adhesion proteins. We transfected a megakaryoblast cell line (Meg-01) to deplete β-dystroglycan, subsequent to their differentiation to the platelet progenitors. Our data showed a direct interaction of the MLR with cytoskeleton to regulate platelet shape, while an association of caveolin-1 PY14 with vinculin is needed to establish focal adhesions, which are modulated for β-dystroglycan. In conclusion, caveolin-1 PY14 in association with platelet cytoskeleton participate in focal adhesions dynamics. © 2015 Wiley Periodicals, Inc.

Revealing the Effects of Nanoscale Membrane Curvature on Lipid Mobility.

PubMed

Kabbani, Abir Maarouf; Woodward, Xinxin; Kelly, Christopher V

2017-10-18

Recent advances in nanoengineering and super-resolution microscopy have enabled new capabilities for creating and observing membrane curvature. However, the effects of curvature on single-lipid diffusion have yet to be revealed. The simulations presented here describe the capabilities of varying experimental methods for revealing the effects of nanoscale curvature on single-molecule mobility. Traditionally, lipid mobility is revealed through fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and single particle tracking (SPT). However, these techniques vary greatly in their ability to detect the effects of nanoscale curvature on lipid behavior. Traditionally, FRAP and FCS depend on diffraction-limited illumination and detection. A simulation of FRAP shows minimal effects on lipids diffusion due to a 50 nm radius membrane bud. Throughout the stages of the budding process, FRAP detected minimal changes in lipid recovery time due to the curvature versus flat membrane. Simulated FCS demonstrated small effects due to a 50 nm radius membrane bud that was more apparent with curvature-dependent lipid mobility changes. However, SPT achieves a sub-diffraction-limited resolution of membrane budding and lipid mobility through the identification of the single-lipid positions with ≤15 nm spatial and ≤20 ms temporal resolution. By mapping the single-lipid step lengths to locations on the membrane, the effects of membrane topography and curvature could be correlated to the effective membrane viscosity. Single-fluorophore localization techniques, such SPT, can detect membrane curvature and its effects on lipid behavior. These simulations and discussion provide a guideline for optimizing the experimental procedures in revealing the effects of curvature on lipid mobility and effective local membrane viscosity.

Revealing the Effects of Nanoscale Membrane Curvature on Lipid Mobility

PubMed Central

Kabbani, Abir Maarouf; Woodward, Xinxin

2017-01-01

Recent advances in nanoengineering and super-resolution microscopy have enabled new capabilities for creating and observing membrane curvature. However, the effects of curvature on single-lipid diffusion have yet to be revealed. The simulations presented here describe the capabilities of varying experimental methods for revealing the effects of nanoscale curvature on single-molecule mobility. Traditionally, lipid mobility is revealed through fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and single particle tracking (SPT). However, these techniques vary greatly in their ability to detect the effects of nanoscale curvature on lipid behavior. Traditionally, FRAP and FCS depend on diffraction-limited illumination and detection. A simulation of FRAP shows minimal effects on lipids diffusion due to a 50 nm radius membrane bud. Throughout the stages of the budding process, FRAP detected minimal changes in lipid recovery time due to the curvature versus flat membrane. Simulated FCS demonstrated small effects due to a 50 nm radius membrane bud that was more apparent with curvature-dependent lipid mobility changes. However, SPT achieves a sub-diffraction-limited resolution of membrane budding and lipid mobility through the identification of the single-lipid positions with ≤15 nm spatial and ≤20 ms temporal resolution. By mapping the single-lipid step lengths to locations on the membrane, the effects of membrane topography and curvature could be correlated to the effective membrane viscosity. Single-fluorophore localization techniques, such SPT, can detect membrane curvature and its effects on lipid behavior. These simulations and discussion provide a guideline for optimizing the experimental procedures in revealing the effects of curvature on lipid mobility and effective local membrane viscosity. PMID:29057801

Lateral Organization of Lipids in Multi-component Liposomes

NASA Astrophysics Data System (ADS)

Ramachandran, Sanoop; Laradji, Mohamed; Sunil Kumar, P. B.

2009-04-01

Inspite of the fluid nature and low elastic modulus, membranes play a crucial role in maintaining the structural integrity of the cell. Recent experiments have challenged the passive nature of the membrane as proposed by the classical fluid mosaic model. Experiments indicate that biomembranes of eukaryotic cells may be laterally organized into small nanoscopic domains, called rafts, which are rich in sphingomyelin and cholesterol. It is largely believed that this in-plane organization is essential for a variety of physiological functions such as signaling, recruitment of specific proteins and endocytosis. However, elucidation of the fundamental issues including the mechanisms leading to the formation of lipid rafts, their stability, and their size remain difficult. This has reiterated the importance of understanding the equilibrium phase behavior and the kinetics of fluid multicomponent lipid membranes before attempts are made to find the effects of more complex mechanisms that may be involved in the formation and stability of lipid rafts. Current increase in interest in the domain formation in multicomponent membranes also stems from the experiments demonstrating fluid-fluid coexistence in mixtures of lipids and cholesterol and the success of several computational models in predicting their behavior. Here we review time dependent Ginzburg Landau model, dynamical triangulation Monte Carlo, and dissipative particle dynamics which are some of the methods that are commonly employed.

Redox signaling via lipid raft clustering in homocysteine-induced injury of podocytes.

PubMed

Zhang, Chun; Hu, Jun-Jun; Xia, Min; Boini, Krishna M; Brimson, Christopher; Li, Pin-Lan

2010-04-01

Our recent studies have indicated that hyperhomocysteinemia (hHcys) may induce podocyte damage, resulting in glomerulosclerosis. However, the molecular mechanisms mediating hHcys-induced podocyte injury are still poorly understood. In the present study, we first demonstrated that an intact NADPH oxidase system is present in podocytes as shown by detection of its membrane subunit (gp91(phox)) and cytosolic subunit (p47(phox)). Then, confocal microscopy showed that gp91(phox) and p47(phox) could be aggregated in lipid raft (LR) clusters in podocytes treated with homocysteine (Hcys), which were illustrated by their colocalization with cholera toxin B, a common LR marker. Different mechanistic LR disruptors, either methyl-beta-cyclodextrin (MCD) or filipin abolished such Hcys-induced formation of LR-gp91(phox) or LR-p47(phox) transmembrane signaling complexes. By flotation of detergent-resistant membrane fractions we found that gp91(phox) and p47(phox) were enriched in LR fractions upon Hcys stimulation, and such enrichment of NADPH oxidase subunits and increase in its enzyme activity were blocked by MCD or filipin. Functionally, disruption of LR clustering significantly attenuated Hcys-induced podocyte injury, as shown by their inhibitory effects on Hcys-decreased expression of slit diaphragm molecules such as nephrin and podocin. Similarly, Hcys-increased expression of desmin was also reduced by disruption of LR clustering. In addition, inhibition of such LR-associated redox signaling prevented cytoskeleton disarrangement and apoptosis induced by Hcys. It is concluded that NADPH oxidase subunits aggregation and consequent activation of this enzyme through LR clustering is an important molecular mechanism triggering oxidative injury of podocytes induced by Hcys. 2009 Elsevier B.V. All rights reserved.

Redox signaling via lipid raft clustering in homocysteine-induced injury of podocytes

PubMed Central

Zhang, Chun; Hu, Jun-Jun; Xia, Min; Boini, Krishna M.; Brimson, Christopher; Li, Pin-Lan

2010-01-01

Our recent studies have indicated that hyperhomocysteinemia (hHcys) may induce podocyte damage, resulting in glomerulosclerosis. However, the molecular mechanisms mediating hHcys-induced podocyte injury are still poorly understood. In the present study, we first demonstrated that an intact NADPH oxidase system is present in podocytes as shown by detection of its membrane subunit (gp91phox) and cytosolic subunit (p47phox). Then, confocal microscopy showed that gp91phox and p47phox could be aggregated in lipid raft (LR) clusters in podocytes treated with homocysteine (Hcys), which were illustrated by their co-localization with cholera toxin B, a common LR marker. Different mechanistic LR disruptors, either methyl-β-cyclodextrin (MCD) or filipin abolished such Hcys-induced formation of LR-gp91phox or LR-p47phox transmembrane signaling complexes. By flotation of detergent-resistant membrane fractions we found that gp91phox and p47phox were enriched in LR fractions upon Hcys stimulation, and such enrichment of NADPH oxidase subunits and increase in its enzyme activity were blocked by MCD or filipin. Functionally, disruption of LR clustering significantly attenuated Hcys-induced podocyte injury, as shown by their inhibitory effects on Hcys-decreased expression of slit diaphragm molecules such as nephrin and podocin. Similarly, Hcys-increased expression of desmin was also reduced by disruption of LR clustering. In addition, inhibition of such LR-associated redox signaling prevented cytoskeleton disarrangement and apoptosis induced by Hcys. It is concluded that NADPH oxidase subunits aggregation and consequent activation of this enzyme through LR clustering is an important molecular mechanism triggering oxidative injury of podocytes induced by Hcys. PMID:20036696

Lipid Rafts and Clathrin Cooperate in the Internalization of PrPC in Epithelial FRT Cells

PubMed Central

Casanova, Philippe; Puri, Claudia; Paladino, Simona; Tivodar, Simona S.; Campana, Vincenza; Tacchetti, Carlo; Zurzolo, Chiara

2009-01-01

Background The cellular prion protein (PrPC) plays a key role in the pathogenesis of Transmissible Spongiform Encephalopathies in which the protein undergoes post-translational conversion to the infectious form (PrPSc). Although endocytosis appears to be required for this conversion, the mechanism of PrPC internalization is still debated, as caveolae/raft- and clathrin-dependent processes have all been reported to be involved. Methodology/Principal Findings We have investigated the mechanism of PrPC endocytosis in Fischer Rat Thyroid (FRT) cells, which lack caveolin-1 (cav-1) and caveolae, and in FRT/cav-1 cells which form functional caveolae. We show that PrPC internalization requires activated Cdc-42 and is sensitive to cholesterol depletion but not to cav-1 expression suggesting a role for rafts but not for caveolae in PrPC endocytosis. PrPC internalization is also affected by knock down of clathrin and by the expression of dominant negative Eps15 and Dynamin 2 mutants, indicating the involvement of a clathrin-dependent pathway. Notably, PrPC co-immunoprecipitates with clathrin and remains associated with detergent-insoluble microdomains during internalization thus indicating that PrPC can enter the cell via multiple pathways and that rafts and clathrin cooperate in its internalization. Conclusions/Significance These findings are of particular interest if we consider that the internalization route/s undertaken by PrPC can be crucial for the ability of different prion strains to infect and to replicate in different cell lines. PMID:19503793

Lipid Clustering Correlates with Membrane Curvature as Revealed by Molecular Simulations of Complex Lipid Bilayers

PubMed Central

Koldsø, Heidi; Shorthouse, David; Hélie, Jean; Sansom, Mark S. P.

2014-01-01

Cell membranes are complex multicomponent systems, which are highly heterogeneous in the lipid distribution and composition. To date, most molecular simulations have focussed on relatively simple lipid compositions, helping to inform our understanding of in vitro experimental studies. Here we describe on simulations of complex asymmetric plasma membrane model, which contains seven different lipids species including the glycolipid GM3 in the outer leaflet and the anionic lipid, phosphatidylinositol 4,5-bisphophate (PIP2), in the inner leaflet. Plasma membrane models consisting of 1500 lipids and resembling the in vivo composition were constructed and simulations were run for 5 µs. In these simulations the most striking feature was the formation of nano-clusters of GM3 within the outer leaflet. In simulations of protein interactions within a plasma membrane model, GM3, PIP2, and cholesterol all formed favorable interactions with the model α-helical protein. A larger scale simulation of a model plasma membrane containing 6000 lipid molecules revealed correlations between curvature of the bilayer surface and clustering of lipid molecules. In particular, the concave (when viewed from the extracellular side) regions of the bilayer surface were locally enriched in GM3. In summary, these simulations explore the nanoscale dynamics of model bilayers which mimic the in vivo lipid composition of mammalian plasma membranes, revealing emergent nanoscale membrane organization which may be coupled both to fluctuations in local membrane geometry and to interactions with proteins. PMID:25340788

Lipid clustering correlates with membrane curvature as revealed by molecular simulations of complex lipid bilayers.

PubMed

Koldsø, Heidi; Shorthouse, David; Hélie, Jean; Sansom, Mark S P

2014-10-01

Cell membranes are complex multicomponent systems, which are highly heterogeneous in the lipid distribution and composition. To date, most molecular simulations have focussed on relatively simple lipid compositions, helping to inform our understanding of in vitro experimental studies. Here we describe on simulations of complex asymmetric plasma membrane model, which contains seven different lipids species including the glycolipid GM3 in the outer leaflet and the anionic lipid, phosphatidylinositol 4,5-bisphophate (PIP2), in the inner leaflet. Plasma membrane models consisting of 1500 lipids and resembling the in vivo composition were constructed and simulations were run for 5 µs. In these simulations the most striking feature was the formation of nano-clusters of GM3 within the outer leaflet. In simulations of protein interactions within a plasma membrane model, GM3, PIP2, and cholesterol all formed favorable interactions with the model α-helical protein. A larger scale simulation of a model plasma membrane containing 6000 lipid molecules revealed correlations between curvature of the bilayer surface and clustering of lipid molecules. In particular, the concave (when viewed from the extracellular side) regions of the bilayer surface were locally enriched in GM3. In summary, these simulations explore the nanoscale dynamics of model bilayers which mimic the in vivo lipid composition of mammalian plasma membranes, revealing emergent nanoscale membrane organization which may be coupled both to fluctuations in local membrane geometry and to interactions with proteins.

Chiral twist drives raft formation and organization in membranes composed of rod-like particles

PubMed Central

Lubensky, Tom C.

2017-01-01

Lipid rafts are hypothesized to facilitate protein interaction, tension regulation, and trafficking in biological membranes, but the mechanisms responsible for their formation and maintenance are not clear. Insights into many other condensed matter phenomena have come from colloidal systems, whose micron-scale particles mimic basic properties of atoms and molecules but permit dynamic visualization with single-particle resolution. Recently, experiments showed that bidisperse mixtures of filamentous viruses can self-assemble into colloidal monolayers with thermodynamically stable rafts exhibiting chiral structure and repulsive interactions. We quantitatively explain these observations by modeling the membrane particles as chiral liquid crystals. Chiral twist promotes the formation of finite-sized rafts and mediates a repulsion that distributes them evenly throughout the membrane. Although this system is composed of filamentous viruses whose aggregation is entropically driven by dextran depletants instead of phospholipids and cholesterol with prominent electrostatic interactions, colloidal and biological membranes share many of the same physical symmetries. Chiral twist can contribute to the behavior of both systems and may account for certain stereospecific effects observed in molecular membranes. PMID:27999184

Sinking a Granular Raft

NASA Astrophysics Data System (ADS)

Protière, Suzie; Josserand, Christophe; Aristoff, Jeffrey M.; Stone, Howard A.; Abkarian, Manouk

2017-03-01

We report experiments that yield new insights on the behavior of granular rafts at an oil-water interface. We show that these particle aggregates can float or sink depending on dimensionless parameters taking into account the particle densities and size and the densities of the two fluids. We characterize the raft shape and stability and propose a model to predict its shape and maximum length to remain afloat. Finally we find that wrinkles and folds appear along the raft due to compression by its own weight, which can trigger destabilization. These features are characteristics of an elastic instability, which we discuss, including the limitations of our model.

Steric Pressure among Membrane-Bound Polymers Opposes Lipid Phase Separation.

PubMed

Imam, Zachary I; Kenyon, Laura E; Carrillo, Adelita; Espinoza, Isai; Nagib, Fatema; Stachowiak, Jeanne C

2016-04-19

Lipid rafts are thought to be key organizers of membrane-protein complexes in cells. Many proteins that interact with rafts have bulky polymeric components such as intrinsically disordered protein domains and polysaccharide chains. Therefore, understanding the interaction between membrane domains and membrane-bound polymers provides insights into the roles rafts play in cells. Multiple studies have demonstrated that high concentrations of membrane-bound polymeric domains create significant lateral steric pressure at membrane surfaces. Furthermore, our recent work has shown that lateral steric pressure at membrane surfaces opposes the assembly of membrane domains. Building on these findings, here we report that membrane-bound polymers are potent suppressors of membrane phase separation, which can destabilize lipid domains with substantially greater efficiency than globular domains such as membrane-bound proteins. Specifically, we created giant vesicles with a ternary lipid composition, which separated into coexisting liquid ordered and disordered phases. Lipids with saturated tails and poly(ethylene glycol) (PEG) chains conjugated to their head groups were included at increasing molar concentrations. When these lipids were sparse on the membrane surface they partitioned to the liquid ordered phase. However, as they became more concentrated, the fraction of GUVs that were phase-separated decreased dramatically, ultimately yielding a population of homogeneous membrane vesicles. Experiments and physical modeling using compositions of increasing PEG molecular weight and lipid miscibility phase transition temperature demonstrate that longer polymers are the most efficient suppressors of membrane phase separation when the energetic barrier to lipid mixing is low. In contrast, as the miscibility transition temperature increases, longer polymers are more readily driven out of domains by the increased steric pressure. Therefore, the concentration of shorter polymers required

Desmosome Assembly and Disassembly Are Membrane Raft-Dependent

PubMed Central

Faundez, Victor; Koval, Michael; Mattheyses, Alexa L.; Kowalczyk, Andrew P.

2014-01-01

Strong intercellular adhesion is critical for tissues that experience mechanical stress, such as the skin and heart. Desmosomes provide adhesive strength to tissues by anchoring desmosomal cadherins of neighboring cells to the intermediate filament cytoskeleton. Alterations in assembly and disassembly compromise desmosome function and may contribute to human diseases, such as the autoimmune skin blistering disease pemphigus vulgaris (PV). We previously demonstrated that PV auto-antibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3) cause loss of adhesion by triggering membrane raft-mediated Dsg3 endocytosis. We hypothesized that raft membrane microdomains play a broader role in desmosome homeostasis by regulating the dynamics of desmosome assembly and disassembly. In human keratinocytes, Dsg3 is raft associated as determined by biochemical and super resolution immunofluorescence microscopy methods. Cholesterol depletion, which disrupts rafts, prevented desmosome assembly and adhesion, thus functionally linking rafts to desmosome formation. Interestingly, Dsg3 did not associate with rafts in cells lacking desmosomal proteins. Additionally, PV IgG-induced desmosome disassembly occurred by redistribution of Dsg3 into raft-containing endocytic membrane domains, resulting in cholesterol-dependent loss of adhesion. These findings demonstrate that membrane rafts are required for desmosome assembly and disassembly dynamics, suggesting therapeutic potential for raft targeting agents in desmosomal diseases such as PV. PMID:24498201

Oceanic rafting by a coastal community

PubMed Central

Fraser, Ceridwen I.; Nikula, Raisa; Waters, Jonathan M.

2011-01-01

Oceanic rafting is thought to play a fundamental role in assembling the biological communities of isolated coastal ecosystems. Direct observations of this key ecological and evolutionary process are, however, critically lacking. The importance of macroalgal rafting as a dispersal mechanism has remained uncertain, largely owing to lack of knowledge about the capacity of fauna to survive long voyages at sea and successfully make landfall and establish. Here, we directly document the rafting of a diverse assemblage of intertidal organisms across several hundred kilometres of open ocean, from the subantarctic to mainland New Zealand. Multispecies analyses using phylogeographic and ecological data indicate that 10 epifaunal invertebrate species rafted on six large bull kelp specimens for several weeks from the subantarctic Auckland and/or Snares Islands to the Otago coast of New Zealand, a minimum distance of some 400–600 km. These genetic data are the first to demonstrate that passive rafting can enable simultaneous trans-oceanic transport and landfall of numerous coastal taxa. PMID:20843850

Oceanic rafting by a coastal community.

PubMed

Fraser, Ceridwen I; Nikula, Raisa; Waters, Jonathan M

2011-03-07

Oceanic rafting is thought to play a fundamental role in assembling the biological communities of isolated coastal ecosystems. Direct observations of this key ecological and evolutionary process are, however, critically lacking. The importance of macroalgal rafting as a dispersal mechanism has remained uncertain, largely owing to lack of knowledge about the capacity of fauna to survive long voyages at sea and successfully make landfall and establish. Here, we directly document the rafting of a diverse assemblage of intertidal organisms across several hundred kilometres of open ocean, from the subantarctic to mainland New Zealand. Multispecies analyses using phylogeographic and ecological data indicate that 10 epifaunal invertebrate species rafted on six large bull kelp specimens for several weeks from the subantarctic Auckland and/or Snares Islands to the Otago coast of New Zealand, a minimum distance of some 400-600 km. These genetic data are the first to demonstrate that passive rafting can enable simultaneous trans-oceanic transport and landfall of numerous coastal taxa.

Oral PEG 15-20 protects the intestine against radiation : role of lipid rafts.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Valuckaite, V.; Zaborina, O.; Long, J.

Intestinal injury following abdominal radiation therapy or accidental exposure remains a significant clinical problem that can result in varying degrees of mucosal destruction such as ulceration, vascular sclerosis, intestinal wall fibrosis, loss of barrier function, and even lethal gut-derived sepsis. We determined the ability of a high-molecular-weight polyethylene glycol-based copolymer, PEG 15-20, to protect the intestine against the early and late effects of radiation in mice and rats and to determine its mechanism of action by examining cultured rat intestinal epithelia. Rats were exposed to fractionated radiation in an established model of intestinal injury, whereby an intestinal segment is surgicallymore » placed into the scrotum and radiated daily. Radiation injury score was decreased in a dose-dependent manner in rats gavaged with 0.5 or 2.0 g/kg per day of PEG 15-20 (n = 9-13/group, P < 0.005). Complementary studies were performed in a novel mouse model of abdominal radiation followed by intestinal inoculation with Pseudomonas aeruginosa (P. aeruginosa), a common pathogen that causes lethal gut-derived sepsis following radiation. Mice mortality was decreased by 40% in mice drinking 1% PEG 15-20 (n = 10/group, P < 0.001). Parallel studies were performed in cultured rat intestinal epithelial cells treated with PEG 15-20 before radiation. Results demonstrated that PEG 15-20 prevented radiation-induced intestinal injury in rats, prevented apoptosis and lethal sepsis attributable to P. aeruginosa in mice, and protected cultured intestinal epithelial cells from apoptosis and microbial adherence and possible invasion. PEG 15-20 appeared to exert its protective effect via its binding to lipid rafts by preventing their coalescence, a hallmark feature in intestinal epithelial cells exposed to radiation.« less

P2X1 receptors localized in lipid rafts mediate ATP motor responses in the human vas deferens longitudinal muscles.

PubMed

Donoso, María Verónica; Norambuena, Andrés; Navarrete, Camilo; Poblete, Inés; Velasco, Alfredo; Huidobro-Toro, Juan Pablo

2014-02-01

To assess the role of the P2X1 receptors (P2X1R) in the longitudinal and circular layers of the human vas deferens, ex vivo-isolated strips or rings were prepared from tissue biopsies to record isometric contractions. To ascertain its membrane distribution, tissue extracts were analyzed by immunoblotting following sucrose gradient ultracentrifugation. ATP, alpha,beta-methylene ATP, or electrical field stimulation elicited robust contractions of the longitudinal layer but not of the circular layer which demonstrated inconsistent responses. Alpha,beta-methylene ATP generated stronger and more robust contractions than ATP. In parallel, prostatic segments of the rat vas deferens were examined. The motor responses in both species were not sustained but decayed within the first minute, showing desensitization to additional applications. Cross-desensitization was established between alpha,beta-methylene ATP or ATP-evoked contractions and electrical field stimulation-induced contractions. Full recovery of the desensitized motor responses required more than 30 min and showed a similar pattern in human and rat tissues. Immunoblot analysis of the human vas deferens extracts revealed a P2X1R oligomer of approximately 200 kDa under nonreducing conditions, whereas dithiothreitol-treated extracts showed a single band of approximately 70 kDa. The P2X1R was identified in ultracentrifugation fractions containing 15%-29% sucrose; the receptor localized in the same fractions as flotillin-1, indicating that it regionalized into smooth muscle lipid rafts. In conclusion, ATP plays a key role in human vas deferens contractile responses of the longitudinal smooth muscle layer, an effect mediated through P2X1Rs.

The Bile Acid Receptor TGR5 Does Not Interact with β-Arrestins or Traffic to Endosomes but Transmits Sustained Signals from Plasma Membrane Rafts*

PubMed Central

Jensen, Dane D.; Godfrey, Cody B.; Niklas, Christian; Canals, Meritxell; Kocan, Martina; Poole, Daniel P.; Murphy, Jane E.; Alemi, Farzad; Cottrell, Graeme S.; Korbmacher, Christoph; Lambert, Nevin A.; Bunnett, Nigel W.; Corvera, Carlos U.

2013-01-01

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with β-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists

New insights on glucosylated lipids: metabolism and functions.

PubMed

Ishibashi, Yohei; Kohyama-Koganeya, Ayako; Hirabayashi, Yoshio

2013-09-01

Ceramide, cholesterol, and phosphatidic acid are major basic structures for cell membrane lipids. These lipids are modified with glucose to generate glucosylceramide (GlcCer), cholesterylglucoside (ChlGlc), and phosphatidylglucoside (PtdGlc), respectively. Glucosylation dramatically changes the functional properties of lipids. For instance, ceramide acts as a strong tumor suppressor that causes apoptosis and cell cycle arrest, while GlcCer has an opposite effect, downregulating ceramide activities. All glucosylated lipids are enriched in lipid rafts or microdomains and play fundamental roles in a variety of cellular processes. In this review, we discuss the biological functions and metabolism of these three glucosylated lipids. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Manipulating membrane lipid profiles to restore T-cell function in autoimmunity.

PubMed

Waddington, Kirsty E; Jury, Elizabeth C

2015-08-01

Plasma membrane lipid rafts are heterogeneous cholesterol and glycosphingolipid (GSL)-enriched microdomains, within which the tight packing of cholesterol with the saturated-acyl chains of GSLs creates a region of liquid-order relative to the surrounding disordered membrane. Thus lipid rafts govern the lateral mobility and interaction of membrane proteins and regulate a plethora of signal transduction events, including T-cell antigen receptor (TCR) signalling. The pathways regulating homoeostasis of membrane cholesterol and GSLs are tightly controlled and alteration of these metabolic processes coincides with immune cell dysfunction as is evident in atherosclerosis, cancer and autoimmunity. Indeed, membrane lipid composition is emerging as an important factor influencing the ability of cells to respond appropriately to microenvironmental stimuli. Consequently, there is increasing interest in targeting membrane lipids or their metabolic control as a novel therapeutic approach to modulate immune cell behaviour and our recent work demonstrates that this is a promising strategy in T-cells from patients with the autoimmune disease systemic lupus erythematosus (SLE). © 2015 Authors; published by Portland Press Limited.

Dynamics and Size of Cross-Linking-Induced Lipid Nanodomains in Model Membranes

PubMed Central

Štefl, Martin; Šachl, Radek; Humpolíčková, Jana; Cebecauer, Marek; Macháň, Radek; Kolářová, Marie; Johansson, Lennart B.-Å.; Hof, Martin

2012-01-01

Changes of membrane organization upon cross-linking of its components trigger cell signaling response to various exogenous factors. Cross-linking of raft gangliosides GM1 with cholera toxin (CTxB) was shown to cause microscopic phase separation in model membranes, and the CTxB-GM1 complexes forming a minimal lipid raft unit are the subject of ongoing cell membrane research. Yet, those subdiffraction sized rafts have never been described in terms of size and dynamics. By means of two-color z-scan fluorescence correlation spectroscopy, we show that the nanosized domains are formed in model membranes at lower sphingomyelin (Sph) content than needed for the large-scale phase separation and that the CTxB-GM1 complexes are confined in the domains poorly stabilized with Sph. Förster resonance energy transfer together with Monte Carlo modeling of the donor decay response reveal the domain radius of ∼8 nm, which increases at higher Sph content. We observed two types of domains behaving differently, which suggests a dual role of the cross-linker: first, local transient condensation of the GM1 molecules compensating for a lack of Sph and second, coalescence of existing nanodomains ending in large-scale phase separation. PMID:22824274

Complex roles of hybrid lipids in the composition, order, and size of lipid membrane domains.

PubMed

Hassan-Zadeh, Ebrahim; Baykal-Caglar, Eda; Alwarawrah, Mohammad; Huang, Juyang

2014-02-11

Hybrid lipids (HL) are phospholipids with one saturated chain and one unsaturated chain. HL are hypothesized to act as linactants (i.e., 2D surfactants) in cell membranes, reducing line tension and creating nanoscopic lipid domains. Here we compare three hybrid lipids of different chain unsaturation (16:0-18:1PC (POPC), 16:0-18:2PC (PLPC), and 16:0-20:4PC (PAPC)) in their abilities to alter the composition, line tension, order, and compactness of lipid domains. We found that the liquid-ordered (Lo) and liquid-disordered (Ld) lipid domains in PAPC/di18:0PC(DSPC)/cholesterol and PLPC/DSPC/cholesterol mixtures are micrometer-sized, and only the POPC/DSPC/cholesterol system has nanoscopic domains. The results indicate that some HLs with polyunsaturated chains are not linactants, and the monounsaturated POPC displays both properties of weak linactants and "Ld-phase" lipids such as di18:1PC (DOPC). The obtained phase boundaries from giant unilamellar vesicles (GUV) show that both POPC and PLPC partition well in the Lo phases. Our MD simulations reveal that these hybrid lipids decrease the order and compactness of Lo domains. Thus, hybrid lipids distinguish themselves from other lipid groups in this combined "partitioning and loosening" ability, which could explain why the Lo domains of GUVs, which often do not contain HL, are more compact than the raft domains in cell membranes. Our line tension measurement and Monte Carlo simulation both show that even the monounsaturated POPC is a weak linactant with only modest ability to occupy domain boundaries and reduce line tension. A more important property of HLs is that they can reduce physical property differences of Lo and Ld bulk domains, which also reduces line tension at domain boundaries.

RAFT polymerization and some of its applications.

PubMed

Moad, Graeme; Rizzardo, Ezio; Thang, San H

2013-08-01

Reversible addition-fragmentation chain transfer (RAFT) is one of the most robust and versatile methods for controlling radical polymerization. With appropriate selection of the RAFT agent for the monomers and reaction conditions, it is applicable to the majority of monomers subject to radical polymerization. The process can be used in the synthesis of well-defined homo-, gradient, diblock, triblock, and star polymers and more complex architectures, which include microgels and polymer brushes. In this Focus Review we describe how the development of RAFT and RAFT application has been facilitated by the adoption of continuous flow techniques using tubular reactors and through the use of high-throughput methodology. Applications described include the use of RAFT in the preparation of polymers for optoelectronics, block copolymer therapeutics, and star polymer rheology control agents. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cellular convection in a chamber with a warm surface raft

NASA Astrophysics Data System (ADS)

Whitehead, J. A.; Shea, Erin; Behn, Mark D.

2011-10-01

We calculate velocity and temperature fields for Rayleigh-Benard convection in a chamber with a warm raft that floats along the top surface for Rayleigh number up to Ra = 20 000. Two-dimensional, infinite Prandtl number, Boussinesq approximation equations are numerically advanced in time from a motionless state in a chamber of length L' and depth D'. We consider cases with an insulated raft and a raft of fixed temperature. Either oscillatory or stationary flow exists. In the case with an insulated raft over a fluid, there are only three parameters that govern the system: Rayleigh number (Ra), scaled chamber length (L = L'/D'), and scaled raft width (W). For W = 0 and L = 1, linear theory shows that the marginal state without a raft is at a Rayleigh number of 23π4=779.3, but we find that for the smallest W (determined by numerical grid size) the raft approaches the center monotonically in time for Ra<790. For 790raft has a decaying oscillation consisting of raft movement back and forth accompanied by convection cell reversal. For 811raft motion for Ra >871. For larger raft widths, there is a range of W that produces raft oscillation at each Ra up to 20 000. Rafts in longer cavities (L = 2 and 4) have almost no oscillatory behavior. With a raft of temperature set to different values of Tr rather than insulating, a fixed Rayleigh number Ra =20000, a square chamber (L = 1), fixed raft width, and with internal heat generation, there are two ranges of oscillating flow.

αVβ3 Integrin-Targeted Radionuclide Therapy with 64Cu-cyclam-RAFT-c(-RGDfK-)4.

PubMed

Jin, Zhao-Hui; Furukawa, Takako; Degardin, Mélissa; Sugyo, Aya; Tsuji, Atsushi B; Yamasaki, Tomoteru; Kawamura, Kazunori; Fujibayashi, Yasuhisa; Zhang, Ming-Rong; Boturyn, Didier; Dumy, Pascal; Saga, Tsuneo

2016-09-01

The transmembrane cell adhesion receptor αVβ3 integrin (αVβ3) has been identified as an important molecular target for cancer imaging and therapy. We have developed a tetrameric cyclic RGD (Arg-Gly-Asp) peptide-based radiotracer (64)Cu-cyclam-RAFT-c(-RGDfK-)4, which successfully captured αVβ3-positive tumors and angiogenesis by PET. Here, we subsequently evaluated its therapeutic potential and side effects using an established αVβ3-positive tumor mouse model. Mice with subcutaneous U87MG glioblastoma xenografts received single administrations of 37 and 74 MBq of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 (37 MBq/nmol), peptide control, or vehicle solution and underwent tumor growth evaluation. Side effects were assessed in tumor-bearing and tumor-free mice in terms of body weight, routine hematology, and hepatorenal functions. Biodistribution of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 with ascending peptide doses (0.25-10 nmol) and with the therapeutic dose of 2 nmol were determined at 3 hours and at various time points (2 minutes-24 hours) postinjection, respectively, based on which radiation-absorbed doses were estimated. The results revealed that (64)Cu-cyclam-RAFT-c(-RGDfK-)4 dose dependently slowed down the tumor growth. The mean tumor doses were 1.28 and 1.81 Gy from 37 and 74 MBq of (64)Cu-cyclam-RAFT-c(-RGDfK-)4, respectively. Peptide dose study showed that the tumor uptake of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 dose dependently decreased at doses ≥1 nmol, indicating a saturation of αVβ3 with the administered therapeutic doses (1 and 2 nmol). Combined analysis of the data from tumor-bearing and tumor-free mice revealed no significant toxicity caused by 37-74 MBq of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 Our study demonstrates the therapeutic efficacy and safety of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 for αVβ3-targeted radionuclide therapy. (64)Cu-cyclam-RAFT-c(-RGDfK-)4 would be a promising theranostic drug for cancer imaging and therapy. Mol Cancer Ther; 15(9); 2076-85. ©2016 AACR

Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide.

PubMed

Gil, Carles; Dorca-Arévalo, Jonatan; Blasi, Juan

2015-01-01

Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology.

Ant workers exhibit specialization and memory during raft formation.

PubMed

Avril, Amaury; Purcell, Jessica; Chapuisat, Michel

2016-06-01

By working together, social insects achieve tasks that are beyond the reach of single individuals. A striking example of collective behaviour is self-assembly, a process in which individuals link their bodies together to form structures such as chains, ladders, walls or rafts. To get insight into how individual behavioural variation affects the formation of self-assemblages, we investigated the presence of task specialization and the role of past experience in the construction of ant rafts. We subjected groups of Formica selysi workers to two consecutive floods and monitored the position of individuals in rafts. Workers showed specialization in their positions when rafting, with the same individuals consistently occupying the top, middle, base or side position in the raft. The presence of brood modified workers' position and raft shape. Surprisingly, workers' experience in the first rafting trial with brood influenced their behaviour and raft shape in the subsequent trial without brood. Overall, this study sheds light on the importance of workers' specialization and memory in the formation of self-assemblages.

Ant workers exhibit specialization and memory during raft formation

NASA Astrophysics Data System (ADS)

Avril, Amaury; Purcell, Jessica; Chapuisat, Michel

2016-06-01

By working together, social insects achieve tasks that are beyond the reach of single individuals. A striking example of collective behaviour is self-assembly, a process in which individuals link their bodies together to form structures such as chains, ladders, walls or rafts. To get insight into how individual behavioural variation affects the formation of self-assemblages, we investigated the presence of task specialization and the role of past experience in the construction of ant rafts. We subjected groups of Formica selysi workers to two consecutive floods and monitored the position of individuals in rafts. Workers showed specialization in their positions when rafting, with the same individuals consistently occupying the top, middle, base or side position in the raft. The presence of brood modified workers' position and raft shape. Surprisingly, workers' experience in the first rafting trial with brood influenced their behaviour and raft shape in the subsequent trial without brood. Overall, this study sheds light on the importance of workers' specialization and memory in the formation of self-assemblages.

Cellular Convection in a Chamber with a Warm Surface Raft

NASA Astrophysics Data System (ADS)

Whitehead, John; Shea, Erin; Behn, Mark

2011-11-01

We calculate velocity and temperature fields for Rayleigh-Benard convection in a chamber with a warm raft that can float along the top surface for Rayleigh number up to Ra=20,000. Two-dimensional, infinite Prandtl number, Boussinesq approximation equations are numerically advanced in time from a motionless state in a chamber of length L' and depth D'. We consider cases with an insulated raft and a raft of fixed temperature. Either oscillatory or stationary flow exists. The case of an insulated raft has three governing parameters: Ra, scaled chamber length L=L'/D', and scaled raft width W. For W=0 and L=1, the marginal state is at Ra=779.3. For smallest W (determined by numerical grid size) and Ra <790 the raft approaches the center monotonically in time. For 790 raft has a decaying oscillation consisting of raft movement back and forth (and convection cell reversal). For 811 >Ra >871 amplitude is steady, starting small and increasing with larger Ra and for Ra >871 raft movement ceases. For larger W, a range of W and Ra has raft oscillation up to Ra=20,000. Rafts in longer cavities (L=2 and 4) have almost no oscillatory behavior. With a raft of temperature Tr rather than insulating, Ra=20,000, and with internal heating, there are wider ranges of oscillating flow. Thus the presence or absence of motion is very sensitive to W, L, raft thermal properties and Ra. Reasons why are discussed.

Lipids in the cell: organisation regulates function.

PubMed

Santos, Ana L; Preta, Giulio

2018-06-01

Lipids are fundamental building blocks of all cells and play important roles in the pathogenesis of different diseases, including inflammation, autoimmune disease, cancer, and neurodegeneration. The lipid composition of different organelles can vary substantially from cell to cell, but increasing evidence demonstrates that lipids become organised specifically in each compartment, and this organisation is essential for regulating cell function. For example, lipid microdomains in the plasma membrane, known as lipid rafts, are platforms for concentrating protein receptors and can influence intra-cellular signalling. Lipid organisation is tightly regulated and can be observed across different model organisms, including bacteria, yeast, Drosophila, and Caenorhabditis elegans, suggesting that lipid organisation is evolutionarily conserved. In this review, we summarise the importance and function of specific lipid domains in main cellular organelles and discuss recent advances that investigate how these specific and highly regulated structures contribute to diverse biological processes.

Gaussian graphical modeling reveals specific lipid correlations in glioblastoma cells

NASA Astrophysics Data System (ADS)

Mueller, Nikola S.; Krumsiek, Jan; Theis, Fabian J.; Böhm, Christian; Meyer-Bäse, Anke

2011-06-01

Advances in high-throughput measurements of biological specimens necessitate the development of biologically driven computational techniques. To understand the molecular level of many human diseases, such as cancer, lipid quantifications have been shown to offer an excellent opportunity to reveal disease-specific regulations. The data analysis of the cell lipidome, however, remains a challenging task and cannot be accomplished solely based on intuitive reasoning. We have developed a method to identify a lipid correlation network which is entirely disease-specific. A powerful method to correlate experimentally measured lipid levels across the various samples is a Gaussian Graphical Model (GGM), which is based on partial correlation coefficients. In contrast to regular Pearson correlations, partial correlations aim to identify only direct correlations while eliminating indirect associations. Conventional GGM calculations on the entire dataset can, however, not provide information on whether a correlation is truly disease-specific with respect to the disease samples and not a correlation of control samples. Thus, we implemented a novel differential GGM approach unraveling only the disease-specific correlations, and applied it to the lipidome of immortal Glioblastoma tumor cells. A large set of lipid species were measured by mass spectrometry in order to evaluate lipid remodeling as a result to a combination of perturbation of cells inducing programmed cell death, while the other perturbations served solely as biological controls. With the differential GGM, we were able to reveal Glioblastoma-specific lipid correlations to advance biomedical research on novel gene therapies.

Nanoscale Packing Differences in Sphingomyelin and Phosphatidylcholine Revealed by BODIPY Fluorescence in Monolayers: Physiological Implications

PubMed Central

2015-01-01

Phosphatidycholines (PC) with two saturated acyl chains (e.g., dipalmitoyl) mimic natural sphingomyelin (SM) by promoting raft formation in model membranes. However, sphingoid-based lipids, such as SM, rather than saturated-chain PCs have been implicated as key components of lipid rafts in biomembranes. These observations raise questions about the physical packing properties of the phase states that can be formed by these two major plasma membrane lipids with identical phosphocholine headgroups. To investigate, we developed a monolayer platform capable of monitoring changes in surface fluorescence by acquiring multiple spectra during measurement of a lipid force–area isotherm. We relied on the concentration-dependent emission changes of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-labeled PC to detect nanoscale alterations in lipid packing and phase state induced by monolayer lateral compression. The BODIPY-PC probe contained an indacene ring with four symmetrically located methyl (Me) substituents to enhance localization to the lipid hydrocarbon region. Surface fluorescence spectra indicated changes in miscibility even when force–area isotherms showed no deviation from ideal mixing behavior in the surface pressure versus cross-sectional molecular area response. We detected slightly better mixing of Me4-BODIPY-8-PC with the fluid-like, liquid expanded phase of 1-palmitoyl-2-oleoyl-PC compared to N-oleoyl-SM. Remarkably, in the gel-like, liquid condensed phase, Me4-BODIPY-8-PC mixed better with N-palmitoyl-SM than dipalmitoyl-PC, suggesting naturally abundant SMs with saturated acyl chains form gel-like lipid phase(s) with enhanced ability to accommodate deeply embedded components compared to dipalmitoyl-PC gel phase. The findings reveal a fundamental difference in the lateral packing properties of SM and PC that occurs even when their acyl chains match. PMID:24564829

Cholesterol's location in lipid bilayers

DOE PAGES

Marquardt, Drew; Kučerka, Norbert; Wassall, Stephen R.; ...

2016-04-04

It is well known that cholesterol modifies the physical properties of lipid bilayers. For example, the much studied liquid-ordered L o phase contains rapidly diffusing lipids with their acyl chains in the all trans configuration, similar to gel phase bilayers. Moreover, the L o phase is commonly associated with cholesterol-enriched lipid rafts, which are thought to serve as platforms for signaling proteins in the plasma membrane. Cholesterol's location in lipid bilayers has been studied extensively, and it has been shown – at least in some bilayers – to align differently from its canonical upright orientation, where its hydroxyl group ismore » in the vicinity of the lipid–water interface. In this study we review recent works describing cholesterol's location in different model membrane systems with emphasis on results obtained from scattering, spectroscopic and molecular dynamics studies.« less

Radiation-induced controlled polymerization of acrylic acid by RAFT and RAFT-MADIX methods in protic solvents

NASA Astrophysics Data System (ADS)

Sütekin, S. Duygu; Güven, Olgun

2018-01-01

The kinetic investigation of one-pot synthesis of poly(acrylic acid) (PAA) prepared via gamma radiation induced controlled polymerization was reported. PAA homopolymers were prepared by Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization in the presence of trithiocarbonate-based chain transfer agent (CTA) 2-(Dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT) and also by Reversible Addition-Fragmentation/Macromolecular Design by Inter-change of Xanthates (RAFT/MADIX) polymerization in the presence of a xanthate based CTA O-ethyl-S-(1-methoxycarbonyl) ethyl dithiocarbonate (RA1). The polymerizations were performed at room temperature by the virtue of ionizing radiation. Protic solvents were used for the RAFT polymerization of AA considering environmental profits. The linear first-order kinetic plot, close control of molecular weight by the monomer/CTA molar ratio supported that the polymerization proceeds in a living fashion. The linear increase in molecular weight with conversion monitored by Size Exclusion Chromatography (SEC) is another proof of controlling of polymerization. [Monomer]/[RAFT] ratio and conversion was controlled to obtain PAA in the molecular weight range of 6900-35,800 with narrow molecular weight distributions. Reaction kinetics and effect of the amount of RAFT agent were investigated in detail. Between two different types of CTA, trithiocarbonate based DDMAT was found to be more efficient in terms of low dispersity (Đ) and linear first-order kinetic behavior for the radiation induced controlled synthesis of PAA homopolymers.

It’s a lipid’s world: Bioactive lipid metabolism and signaling in neural stem cell differentiation

PubMed Central

Bieberich, Erhard

2012-01-01

Lipids are often considered membrane components whose function is to embed proteins into cell membranes. In the last two decades, studies on brain lipids have unequivocally demonstrated that many lipids have critical cell signaling functions; they are called “bioactive lipids”. Pioneering work in Dr. Robert Ledeen’s laboratory has shown that two bioactive brain sphingolipids, sphingomyelin and the ganglioside GM1 are major signaling lipids in the nuclear envelope. In addition to derivatives of the sphingolipid ceramide, the bioactive lipids discussed here belong to the classes of terpenoids and steroids, eicosanoids, and lysophospholipids. These lipids act mainly through two mechanisms: 1) direct interaction between the bioactive lipid and a specific protein binding partner such as a lipid receptor, protein kinase or phosphatase, ion exchanger, or other cell signaling protein; and 2) formation of lipid microdomains or rafts that regulate the activity of a group of raft-associated cell signaling proteins. In recent years, a third mechanism has emerged, which invokes lipid second messengers as a regulator for the energy and redox balance of differentiating neural stem cells (NSCs). Interestingly, developmental niches such as the stem cell niche for adult NSC differentiation may also be metabolic compartments that respond to a distinct combination of bioactive lipids. The biological function of these lipids as regulators of NSC differentiation will be reviewed and their application in stem cell therapy discussed. PMID:22246226

Colorado Outward Bound School Rafting Manual.

ERIC Educational Resources Information Center

Brown, Al

River rafting trips at the Colorado Outward Bound School (COBS) present participants with an opportunity for developing self-confidence, self-awareness, and concern for others through challenging and adventuresome group effort, combined with a program of instruction in rafting skills, safety consciousness, and awareness of the natural environment.…

Lipidomics of Candida albicans biofilms reveals phase-dependent production of phospholipid molecular classes and role for lipid rafts in biofilm formation.

PubMed

Lattif, Ali Abdul; Mukherjee, Pranab K; Chandra, Jyotsna; Roth, Mary R; Welti, Ruth; Rouabhia, Mahmoud; Ghannoum, Mahmoud A

2011-11-01

Candida albicans-associated bloodstream infections are linked to the ability of this yeast to form biofilms. In this study, we used lipidomics to compare the lipid profiles of C. albicans biofilms and planktonic cells, in early and mature developmental phases. Our results showed that significant differences exist in lipid composition in both developmental phases. Biofilms contained higher levels of phospholipid and sphingolipids than planktonic cells (nmol per g biomass, P<0.05 for all comparisons). In the early phase, levels of lipid in most classes were significantly higher in biofilms compared to planktonic cells (P≤0.05). The ratio of phosphatidylcholine to phosphatidylethanolamine was lower in biofilms compared to planktonic cells in both early (1.17 vs 2.52, P≤0.001) and late (2.34 vs 3.81, P≤0.001) developmental phases. The unsaturation index of phospholipids decreased with time, with this effect being particularly strong for biofilms. Inhibition of the biosynthetic pathway for sphingolipid [mannosyl diinositolphosphoryl ceramide, M(IP)₂C] by myriocin or aureobasidin A, and disruption of the gene encoding inositolphosphotransferase (Ipt1p), abrogated the ability of C. albicans to form biofilms. The differences in lipid profiles between biofilms and planktonic Candida cells may have important implications for the biology and antifungal resistance of biofilms.

Topologically Diverse Human Membrane Proteins Partition to Liquid-Disordered Domains in Phase-Separated Lipid Vesicles.

PubMed

Schlebach, Jonathan P; Barrett, Paul J; Day, Charles A; Kim, Ji Hun; Kenworthy, Anne K; Sanders, Charles R

2016-02-23

The integration of membrane proteins into "lipid raft" membrane domains influences many biochemical processes. The intrinsic structural properties of membrane proteins are thought to mediate their partitioning between membrane domains. However, whether membrane topology influences the targeting of proteins to rafts remains unclear. To address this question, we examined the domain preference of three putative raft-associated membrane proteins with widely different topologies: human caveolin-3, C99 (the 99 residue C-terminal domain of the amyloid precursor protein), and peripheral myelin protein 22. We find that each of these proteins are excluded from the ordered domains of giant unilamellar vesicles containing coexisting liquid-ordered and liquid-disordered phases. Thus, the intrinsic structural properties of these three topologically distinct disease-linked proteins are insufficient to confer affinity for synthetic raft-like domains.

Accelerated Combinatorial High Throughput Star Polymer Synthesis via a Rapid One-Pot Sequential Aqueous RAFT (rosa-RAFT) Polymerization Scheme.

PubMed

Cosson, Steffen; Danial, Maarten; Saint-Amans, Julien Rosselgong; Cooper-White, Justin J

2017-04-01

Advanced polymerization methodologies, such as reversible addition-fragmentation transfer (RAFT), allow unprecedented control over star polymer composition, topology, and functionality. However, using RAFT to produce high throughput (HTP) combinatorial star polymer libraries remains, to date, impracticable due to several technical limitations. Herein, the methodology "rapid one-pot sequential aqueous RAFT" or "rosa-RAFT," in which well-defined homo-, copolymer, and mikto-arm star polymers can be prepared in very low to medium reaction volumes (50 µL to 2 mL) via an "arm-first" approach in air within minutes, is reported. Due to the high conversion of a variety of acrylamide/acrylate monomers achieved during each successive short reaction step (each taking 3 min), the requirement for intermediary purification is avoided, drastically facilitating and accelerating the star synthesis process. The presented methodology enables RAFT to be applied to HTP polymeric bio/nanomaterials discovery pipelines, in which hundreds of complex polymeric formulations can be rapidly produced, screened, and scaled up for assessment in a wide range of applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modification of one man life raft

NASA Technical Reports Server (NTRS)

Soter, E. J. (Inventor)

1974-01-01

A one man inflatable life raft is described. The raft has an inflatable tube perimetrically bounding the occupant receiving space with a flexible floor member. A zippered opening in the floor allows entry and facilitates the use of a constant diameter tube. An airtight fabric bulkhead divides the peripheral tube longitudinally into inflatable tube sections, where if either tube section were punctured, the bulkhead would move into the punctured section to substitute for the punctured wall portion and maintain the inflatable volume of the tube. The floor member is attached to the central portion of the tube wall so that either side of the raft can be the up side.

The Recycling Endosome of Madin-Darby Canine Kidney Cells Is a Mildly Acidic Compartment Rich in Raft Components

PubMed Central

Gagescu, Raluca; Demaurex, Nicolas; Parton, Robert G.; Hunziker, Walter; Huber, Lukas A.; Gruenberg, Jean

2000-01-01

We present a biochemical and morphological characterization of recycling endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes are distinct from conventional early endosomes. We also find that recycling endosomes are less acidic than early endosomes because they lack a functional vacuolar ATPase. Furthermore, we show that recycling endosomes can be reached by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling endosomes are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observations may suggest that a lipid-based sorting mechanism operates along the Madin-Darby canine kidney recycling pathway, contributing to the maintenance of cell polarity. Altogether, our data indicate that recycling endosomes and early endosomes differ functionally and biochemically and thus that different molecular mechanisms regulate protein sorting and membrane traffic at each step of the receptor recycling pathway. PMID:10930469

Compositional changes in lipid microdomains of air-blood barrier plasma membranes in pulmonary interstitial edema.

PubMed

Palestini, Paola; Calvi, Chiara; Conforti, Elena; Daffara, Rossella; Botto, Laura; Miserocchi, Giuseppe

2003-10-01

We evaluated in anesthetized rabbits the compositional changes of plasmalemmal lipid microdomains from lung tissue samples after inducing pulmonary interstitial edema (0.5 ml/kg for 3 h, leading to approximately 5% increase in extravascular water). Lipid microdomains (lipid rafts and caveolae) were present in the detergent-resistant fraction (DRF) obtained after discontinuous sucrose density gradient. DRF was enriched in caveolin-1, flotillin, aquaporin-1, GM1, cholesterol, sphingomyelin, and phosphatidylserine, and their contents significantly increased in interstitial edema. The higher DRF content in caveolin, flotillin, and aquaporin-1 and of the ganglioside GM1 suggests an increase both in caveolar domains and in lipid rafts, respectively. Compositional changes could be ascribed to endothelial and epithelial cells that provide most of plasma membrane surface area in the air-blood barrier. Alterations in lipid components in the plasma membrane may reflect rearrangement of floating lipid platforms within the membrane and/or lipid translocation from intracellular stores. Lipid traffic could be stimulated by the marked increase in hydraulic interstitial pressure after initial water accumulation, from approximately -10 to 5 cmH2O, due to the low compliance of the pulmonary tissue, in particular in the basement membranes and in the interfibrillar substance. Compositional changes in lipid microdomains represent a sign of cellular activation and suggest the potential role of mechanotransduction in response to developing interstitial edema.

Lipoprotein lipase-dependent binding and uptake of low density lipoproteins by THP-1 monocytes and macrophages: possible involvement of lipid rafts.

PubMed

Makoveichuk, Elena; Castel, Susanna; Vilaró, Senen; Olivecrona, Gunilla

2004-11-08

Lipoprotein lipase (LPL) is produced by cells in the artery wall and can mediate binding of lipoproteins to cell surface heparan sulfate proteoglycans (HSPG), resulting in endocytosis (the bridging function). Active, dimeric LPL may dissociate to inactive monomers, the main form found in plasma. We have studied binding/internalization of human low density lipoprotein (LDL), mediated by bovine LPL, using THP-1 monocytes and macrophages. Uptake of (125)I-LDL was similar in monocytes and macrophages and was not affected by the LDL-receptor family antagonist receptor-associated protein (RAP) or by the phagocytosis inhibitor cytochalasin D. In contrast, uptake depended on HSPG and on membrane cholesterol. Incubation in the presence of dexamethasone increased the endogenous production of LPL by the cells and also increased LPL-mediated binding of LDL to the cell surfaces. Monomeric LPL was bound to the cells mostly in a heparin-resistant fashion. We conclude that the uptake of LDL mediated by LPL dimers is receptor-independent and involves cholesterol-enriched membrane areas (lipid rafts). Dimeric and monomeric LPL differ in their ability to mediate binding/uptake of LDL, probably due to different mechanisms for binding/internalization.

Regulation of T-cell receptor signalling by membrane microdomains

PubMed Central

Razzaq, Tahir M; Ozegbe, Patricia; Jury, Elizabeth C; Sembi, Phupinder; Blackwell, Nathan M; Kabouridis, Panagiotis S

2004-01-01

There is now considerable evidence suggesting that the plasma membrane of mammalian cells is compartmentalized by functional lipid raft microdomains. These structures are assemblies of specialized lipids and proteins and have been implicated in diverse biological functions. Analysis of their protein content using proteomics and other methods revealed enrichment of signalling proteins, suggesting a role for these domains in intracellular signalling. In T lymphocytes, structure/function experiments and complementary pharmacological studies have shown that raft microdomains control the localization and function of proteins which are components of signalling pathways regulated by the T-cell antigen receptor (TCR). Based on these studies, a model for TCR phosphorylation in lipid rafts is presented. However, despite substantial progress in the field, critical questions remain. For example, it is unclear if membrane rafts represent a homogeneous population and if their structure is modified upon TCR stimulation. In the future, proteomics and the parallel development of complementary analytical methods will undoubtedly contribute in further delineating the role of lipid rafts in signal transduction mechanisms. PMID:15554919

Wrinkles, folds, and plasticity in granular rafts

NASA Astrophysics Data System (ADS)

Jambon-Puillet, Etienne; Josserand, Christophe; Protière, Suzie

2017-09-01

We investigate the mechanical response of a compressed monolayer of large and dense particles at a liquid-fluid interface: a granular raft. Upon compression, rafts first wrinkle; then, as the confinement increases, the deformation localizes in a unique fold. This characteristic buckling pattern is usually associated with floating elastic sheets, and as a result, particle laden interfaces are often modeled as such. Here, we push this analogy to its limits by comparing quantitative measurements of the raft morphology to a theoretical continuous elastic model of the interface. We show that, although powerful to describe the wrinkle wavelength, the wrinkle-to-fold transition, and the fold shape, this elastic description does not capture the finer details of the experiment. We describe an unpredicted secondary wavelength, a compression discrepancy with the model, and a hysteretic behavior during compression cycles, all of which are a signature of the intrinsic discrete and frictional nature of granular rafts. It suggests also that these composite materials exhibit both plastic transition and jamming dynamics.

Polyunsaturated Fatty Acids in Lipid Bilayers and Tubules

NASA Astrophysics Data System (ADS)

Hirst, Linda S.; Yuan, Jing; Pramudya, Yohannes; Nguyen, Lam T.

2007-03-01

Omega-3 polyunsaturated fatty acids (PUFAs) are found in a variety of biological membranes and have been implicated with lipid raft formation and possible function, typical molecules include DHA (Docosahexanoic Acid) and AA (Alphalinoleic Acid) which have been the focus of considerable attention in recent years. We are interested in the phase behavior of these molecules in the lipid bilayer. The addition of lipid molecules with polyunsaturated chains has a clear effect on the fluidity and curvature of the membrane and we investigate the effects the addition of polyunsaturated lipids on bilayer structure and tubule formation. Self-assembled cylindrical lipid tubules have attracted considerable attention because of their interesting structures and potential technological applications. Using x-ray diffraction techniques, Atomic Force Microscopy and confocal fluorescence imaging, both symmetric and mixed chain lipids were incorporated into model membranes and the effects on bilayer structure and tubule formation investigated.

Generation of organotypic raft cultures from primary human keratinocytes.

PubMed

Anacker, Daniel; Moody, Cary

2012-02-22

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)(1). The life cycle of HPV is tightly linked to the differentiation of squamous epithelium(2). Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production(3,4,5). In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras(6) and modified by Kopan et al.(7), the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies(8). Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as

Niemann-Pick type C proteins promote microautophagy by expanding raft-like membrane domains in the yeast vacuole

PubMed Central

Tsuji, Takuma; Fujimoto, Megumi; Tatematsu, Tsuyako; Cheng, Jinglei; Orii, Minami; Takatori, Sho; Fujimoto, Toyoshi

2017-01-01

Niemann-Pick type C is a storage disease caused by dysfunction of NPC proteins, which transport cholesterol from the lumen of lysosomes to the limiting membrane of that compartment. Using freeze fracture electron microscopy, we show here that the yeast NPC orthologs, Ncr1p and Npc2p, are essential for formation and expansion of raft-like domains in the vacuolar (lysosome) membrane, both in stationary phase and in acute nitrogen starvation. Moreover, the expanded raft-like domains engulf lipid droplets by a microautophagic mechanism. We also found that the multivesicular body pathway plays a crucial role in microautophagy in acute nitrogen starvation by delivering sterol to the vacuole. These data show that NPC proteins promote microautophagy in stationary phase and under nitrogen starvation conditions, likely by increasing sterol in the limiting membrane of the vacuole. DOI: http://dx.doi.org/10.7554/eLife.25960.001 PMID:28590904

Biomedical applications of polymers derived by reversible addition - fragmentation chain-transfer (RAFT).

PubMed

Fairbanks, Benjamin D; Gunatillake, Pathiraja A; Meagher, Laurence

2015-08-30

RAFT- mediated polymerization, providing control over polymer length and architecture as well as facilitating post polymerization modification of end groups, has been applied to virtually every facet of biomedical materials research. RAFT polymers have seen particularly extensive use in drug delivery research. Facile generation of functional and telechelic polymers permits straightforward conjugation to many therapeutic compounds while synthesis of amphiphilic block copolymers via RAFT allows for the generation of self-assembled structures capable of carrying therapeutic payloads. With the large and growing body of literature employing RAFT polymers as drug delivery aids and vehicles, concern over the potential toxicity of RAFT derived polymers has been raised. While literature exploring this complication is relatively limited, the emerging consensus may be summed up in three parts: toxicity of polymers generated with dithiobenzoate RAFT agents is observed at high concentrations but not with polymers generated with trithiocarbonate RAFT agents; even for polymers generated with dithiobenzoate RAFT agents, most reported applications call for concentrations well below the toxicity threshold; and RAFT end-groups may be easily removed via any of a variety of techniques that leave the polymer with no intrinsic toxicity attributable to the mechanism of polymerization. The low toxicity of RAFT-derived polymers and the ability to remove end groups via straightforward and scalable processes make RAFT technology a valuable tool for practically any application in which a polymer of defined molecular weight and architecture is desired. Copyright © 2015. Published by Elsevier B.V.

Lipid profiling of the Arabidopsis hypersensitive response reveals specific lipid peroxidation and fragmentation processes: biogenesis of pimelic and azelaic acid.

PubMed

Zoeller, Maria; Stingl, Nadja; Krischke, Markus; Fekete, Agnes; Waller, Frank; Berger, Susanne; Mueller, Martin J

2012-09-01

Lipid peroxidation (LPO) is induced by a variety of abiotic and biotic stresses. Although LPO is involved in diverse signaling processes, little is known about the oxidation mechanisms and major lipid targets. A systematic lipidomics analysis of LPO in the interaction of Arabidopsis (Arabidopsis thaliana) with Pseudomonas syringae revealed that LPO is predominantly confined to plastid lipids comprising galactolipid and triacylglyceride species and precedes programmed cell death. Singlet oxygen was identified as the major cause of lipid oxidation under basal conditions, while a 13-lipoxygenase (LOX2) and free radical-catalyzed lipid oxidation substantially contribute to the increase upon pathogen infection. Analysis of lox2 mutants revealed that LOX2 is essential for enzymatic membrane peroxidation but not for the pathogen-induced free jasmonate production. Despite massive oxidative modification of plastid lipids, levels of nonoxidized lipids dramatically increased after infection. Pathogen infection also induced an accumulation of fragmented lipids. Analysis of mutants defective in 9-lipoxygenases and LOX2 showed that galactolipid fragmentation is independent of LOXs. We provide strong in vivo evidence for a free radical-catalyzed galactolipid fragmentation mechanism responsible for the formation of the essential biotin precursor pimelic acid as well as of azelaic acid, which was previously postulated to prime the immune response of Arabidopsis. Our results suggest that azelaic acid is a general marker for LPO rather than a general immune signal. The proposed fragmentation mechanism rationalizes the pathogen-induced radical amplification and formation of electrophile signals such as phytoprostanes, malondialdehyde, and hexenal in plastids.

Uptake and intracellular fate of cholera toxin subunit b-modified mesoporous silica nanoparticle-supported lipid bilayers (aka protocells) in motoneurons.

PubMed

Gonzalez Porras, Maria A; Durfee, Paul; Giambini, Sebastian; Sieck, Gary C; Brinker, C Jeffrey; Mantilla, Carlos B

2018-04-01

Cholera toxin B (CTB) modified mesoporous silica nanoparticle supported lipid bilayers (CTB-protocells) are a promising, customizable approach for targeting therapeutic cargo to motoneurons. In the present study, the endocytic mechanism and intracellular fate of CTB-protocells in motoneurons were examined to provide information for the development of therapeutic application and cargo delivery. Pharmacological inhibitors elucidated CTB-protocells endocytosis to be dependent on the integrity of lipid rafts and macropinocytosis. Using immunofluorescence techniques, live confocal and transmission electron microscopy, CTB-protocells were primarily found in the cytosol, membrane lipid domains and Golgi. There was no difference in the amount of motoneuron activity dependent uptake of CTB-protocells in neuromuscular junctions, consistent with clathrin activation at the axon terminals during low frequency activity. In conclusion, CTB-protocells uptake is mediated principally by lipid rafts and macropinocytosis. Once internalized, CTB-protocells escape lysosomal degradation, and engage biological pathways that are not readily accessible by untargeted delivery methods. Copyright © 2018 Elsevier Inc. All rights reserved.

Stability and chaotification of vibration isolation floating raft systems with time-delayed feedback control.

PubMed

Li, Y L; Xu, D L; Fu, Y M; Zhou, J X

2011-09-01

This paper presents a systematic study on the stability of a two-dimensional vibration isolation floating raft system with a time-delayed feedback control. Based on the generalized Sturm criterion, the critical control gain for the delay-independent stability region and critical time delays for the stability switches are derived. The critical conditions can provide a theoretical guidance of chaotification design for line spectra reduction. Numerical simulations verify the correctness of the approach. Bifurcation analyses reveal that chaotification is more likely to occur in unstable region defined by these critical conditions, and the stiffness of the floating raft and mass ratio are the sensitive parameters to reduce critical control gain.

Erythropoietin Receptor Signaling Is Membrane Raft Dependent

PubMed Central

McGraw, Kathy L.; Fuhler, Gwenny M.; Johnson, Joseph O.; Clark, Justine A.; Caceres, Gisela C.; Sokol, Lubomir; List, Alan F.

2012-01-01

Upon erythropoietin (Epo) engagement, Epo-receptor (R) homodimerizes to activate JAK2 and Lyn, which phosphorylate STAT5. Although recent investigations have identified key negative regulators of Epo-R signaling, little is known about the role of membrane localization in controlling receptor signal fidelity. Here we show a critical role for membrane raft (MR) microdomains in creation of discrete signaling platforms essential for Epo-R signaling. Treatment of UT7 cells with Epo induced MR assembly and coalescence. Confocal microscopy showed that raft aggregates significantly increased after Epo stimulation (mean, 4.3±1.4(SE) vs. 25.6±3.2 aggregates/cell; p≤0.001), accompanied by a >3-fold increase in cluster size (p≤0.001). Raft fraction immunoblotting showed Epo-R translocation to MR after Epo stimulation and was confirmed by fluorescence microscopy in Epo stimulated UT7 cells and primary erythroid bursts. Receptor recruitment into MR was accompanied by incorporation of JAK2, Lyn, and STAT5 and their activated forms. Raft disruption by cholesterol depletion extinguished Epo induced Jak2, STAT5, Akt and MAPK phosphorylation in UT7 cells and erythroid progenitors. Furthermore, inhibition of the Rho GTPases Rac1 or RhoA blocked receptor recruitment into raft fractions, indicating a role for these GTPases in receptor trafficking. These data establish a critical role for MR in recruitment and assembly of Epo-R and signal intermediates into discrete membrane signaling units. PMID:22509308

Research status of wave energy conversion (WEC) device of raft structure

NASA Astrophysics Data System (ADS)

Dong, Jianguo; Gao, Jingwei; Tao, Liang; Zheng, Peng

2017-10-01

This paper has briefly described the concept of wave energy generation and six typical conversion devices. As for raft structure, detailed analysis is provided from its development process to typical devices. Taking the design process and working principle of Plamis as an example, the general principle of raft structure is briefly described. After that, a variety of raft structure models are introduced. Finally, the advantages and disadvantages, and development trend of raft structure are pointed out.

Size and mobility of lipid domains tuned by geometrical constraints.

PubMed

Schütte, Ole M; Mey, Ingo; Enderlein, Jörg; Savić, Filip; Geil, Burkhard; Janshoff, Andreas; Steinem, Claudia

2017-07-25

In the plasma membrane of eukaryotic cells, proteins and lipids are organized in clusters, the latter ones often called lipid domains or "lipid rafts." Recent findings highlight the dynamic nature of such domains and the key role of membrane geometry and spatial boundaries. In this study, we used porous substrates with different pore radii to address precisely the extent of the geometric constraint, permitting us to modulate and investigate the size and mobility of lipid domains in phase-separated continuous pore-spanning membranes (PSMs). Fluorescence video microscopy revealed two types of liquid-ordered ( l o ) domains in the freestanding parts of the PSMs: ( i ) immobile domains that were attached to the pore rims and ( ii ) mobile, round-shaped l o domains within the center of the PSMs. Analysis of the diffusion of the mobile l o domains by video microscopy and particle tracking showed that the domains' mobility is slowed down by orders of magnitude compared with the unrestricted case. We attribute the reduced mobility to the geometric confinement of the PSM, because the drag force is increased substantially due to hydrodynamic effects generated by the presence of these boundaries. Our system can serve as an experimental test bed for diffusion of 2D objects in confined geometry. The impact of hydrodynamics on the mobility of enclosed lipid domains can have great implications for the formation and lateral transport of signaling platforms.

Size and mobility of lipid domains tuned by geometrical constraints

PubMed Central

Schütte, Ole M.; Mey, Ingo; Savić, Filip; Geil, Burkhard; Janshoff, Andreas

2017-01-01

In the plasma membrane of eukaryotic cells, proteins and lipids are organized in clusters, the latter ones often called lipid domains or “lipid rafts.” Recent findings highlight the dynamic nature of such domains and the key role of membrane geometry and spatial boundaries. In this study, we used porous substrates with different pore radii to address precisely the extent of the geometric constraint, permitting us to modulate and investigate the size and mobility of lipid domains in phase-separated continuous pore-spanning membranes (PSMs). Fluorescence video microscopy revealed two types of liquid-ordered (lo) domains in the freestanding parts of the PSMs: (i) immobile domains that were attached to the pore rims and (ii) mobile, round-shaped lo domains within the center of the PSMs. Analysis of the diffusion of the mobile lo domains by video microscopy and particle tracking showed that the domains’ mobility is slowed down by orders of magnitude compared with the unrestricted case. We attribute the reduced mobility to the geometric confinement of the PSM, because the drag force is increased substantially due to hydrodynamic effects generated by the presence of these boundaries. Our system can serve as an experimental test bed for diffusion of 2D objects in confined geometry. The impact of hydrodynamics on the mobility of enclosed lipid domains can have great implications for the formation and lateral transport of signaling platforms. PMID:28696315

Formulation and optimisation of raft-forming chewable tablets containing H2 antagonist.

PubMed

Prajapati, Shailesh T; Mehta, Anant P; Modhia, Ishan P; Patel, Chhagan N

2012-10-01

The purpose of this research work was to formulate raft-forming chewable tablets of H2 antagonist (Famotidine) using a raft-forming agent along with an antacid- and gas-generating agent. Tablets were prepared by wet granulation and evaluated for raft strength, acid neutralisation capacity, weight variation, % drug content, thickness, hardness, friability and in vitro drug release. Various raft-forming agents were used in preliminary screening. A 2(3) full-factorial design was used in the present study for optimisation. The amount of sodium alginate, amount of calcium carbonate and amount sodium bicarbonate were selected as independent variables. Raft strength, acid neutralisation capacity and drug release at 30 min were selected as responses. Tablets containing sodium alginate were having maximum raft strength as compared with other raft-forming agents. Acid neutralisation capacity and in vitro drug release of all factorial batches were found to be satisfactory. The F5 batch was optimised based on maximum raft strength and good acid neutralisation capacity. Drug-excipient compatibility study showed no interaction between the drug and excipients. Stability study of the optimised formulation showed that the tablets were stable at accelerated environmental conditions. It was concluded that raft-forming chewable tablets prepared using an optimum amount of sodium alginate, calcium carbonate and sodium bicarbonate could be an efficient dosage form in the treatment of gastro oesophageal reflux disease.

Well-Defined Macromolecules Using Horseradish Peroxidase as a RAFT Initiase.

PubMed

Danielson, Alex P; Bailey-Van Kuren, Dylan; Lucius, Melissa E; Makaroff, Katherine; Williams, Cameron; Page, Richard C; Berberich, Jason A; Konkolewicz, Dominik

2016-02-01

Enzymatic catalysis and control over macromolecular architectures from reversible addition-fragmentation chain transfer polymerization (RAFT) are combined to give a new method of making polymers. Horseradish peroxidase (HRP) is used to catalytically generate radicals using hydrogen peroxide and acetylacetone as a mediator. RAFT is used to control the polymer structure. HRP catalyzed RAFT polymerization gives acrylate and acrylamide polymers with relatively narrow molecular weight distributions. The polymerization is rapid, typically exceeding 90% monomer conversion in 30 min. Complex macromolecular architectures including a block copolymer and a protein-polymer conjugate are synthesized using HRP to catalytically initiate RAFT polymerization. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The raft foundation reinforcement construction technology of Hongyun Building B tower

NASA Astrophysics Data System (ADS)

Liu, Yu; Yin, Suhua; Wu, Yanli; Zhao, Ying

2017-08-01

The foundation of Hongyun building B tower is made of raft board foundation which is 3300mm in the thickness include four kinds of reinforcement Φ32, Φ28, Φ12 and 12 steel grade two, in respective. It is researched that the raft foundation mass concrete construction technology is expatiated from temperature and cracks of the raft foundation and the temperature control and monitoring of the concrete base slab construction and concrete curing. According to the characteristics with large volume and thickness of the engineering of raft foundation, the construction of the reinforced force was calculated and the quality control measures were used to the reinforcement binding and connection, so it is success that Hongyun Building B tower raft foundation reinforced construction.

Manipulating lipid membrane architecture by liquid crystal-analog curvature elasticity (Presentation Recording)

NASA Astrophysics Data System (ADS)

Lee, Sin-Doo

2015-10-01

Soft matters such as liquid crystals and biological molecules exhibit a variety of interesting physical phenomena as well as new applications. Recently, in mimicking biological systems that have the ability to sense, regulate, grow, react, and regenerate in a highly responsive and self-adaptive manner, the significance of the liquid crystal order in living organisms, for example, a biological membrane possessing the lamellar order, is widely recognized from the viewpoints of physics and chemistry of interfaces and membrane biophysics. Lipid bilayers, resembling cell membranes, provide primary functions for the transport of biological components of ions and molecules in various cellular activities, including vesicle budding and membrane fusion, through lateral organization of the membrane components such as proteins. In this lecture, I will describe how the liquid crystal-analog curvature elasticity of a lipid bilayer plays a critical role in developing a new platform for understanding diverse biological functions at a cellular level. The key concept is to manipulate the local curvature at an interface between a solid substrate and a model membrane. Two representative examples will be demonstrated: one of them is the topographic control of lipid rafts in a combinatorial array where the ligand-receptor binding event occurs and the other concerns the reconstitution of a ring-type lipid raft in bud-mimicking architecture within the framework of the curvature elasticity.

Stable, inflatable life raft for high seas rescue operations

NASA Technical Reports Server (NTRS)

Barnett, J. H., Jr.; Harrison, F.; Marak, R.; Radnofsky, M. I.

1971-01-01

Raft is easily deployed and highly maneuverable in water. It has false bottom of water ballast containers attached to underside, making it exceptionally stable platform from which swimmers can operate. Raft is attachable to external moorings.

Super-resolution microscopy reveals the insulin-resistance-regulated reorganization of GLUT4 on plasma membranes.

PubMed

Gao, Lan; Chen, Junling; Gao, Jing; Wang, Hongda; Xiong, Wenyong

2017-01-15

GLUT4 (also known as SLC2A4) is essential for glucose uptake in skeletal muscles and adipocytes, which play central roles in whole-body glucose metabolism. Here, using direct stochastic optical reconstruction microscopy (dSTORM) to investigate the characteristics of plasma-membrane-fused GLUT4 at the single-molecule level, we have demonstrated that insulin and insulin resistance regulate the spatial organization of GLUT4 in adipocytes. Stimulation with insulin shifted the balance of GLUT4 on the plasma membrane toward a more dispersed configuration. In contrast, insulin resistance induced a more clustered distribution of GLUT4 and increased the mean number of molecules per cluster. Furthermore, our data demonstrate that the F 5 QQI motif and lipid rafts mediate the maintenance of GLUT4 clusters on the plasma membrane. Mutation of F 5 QQI (F 5 QQA-GLUT4) induced a more clustered distribution of GLUT4; moreover, destruction of lipid rafts in adipocytes expressing F 5 QQA-GLUT4 dramatically decreased the percentage of large clusters and the mean number of molecules per cluster. In conclusion, our data clarify the effects of insulin stimulation or insulin resistance on GLUT4 reorganization on the plasma membrane and reveal new pathogenic mechanisms of insulin resistance. © 2017. Published by The Company of Biologists Ltd.

Plasma lipidomics reveals potential lipid markers of major depressive disorder.

PubMed

Liu, Xinyu; Li, Jia; Zheng, Peng; Zhao, Xinjie; Zhou, Chanjuan; Hu, Chunxiu; Hou, Xiaoli; Wang, Haiyang; Xie, Peng; Xu, Guowang

2016-09-01

Major depressive disorder (MDD) is a grave debilitating mental disease with a high incidence and severely impairs quality of life. Therefore, its physiopathological basis study and diagnostic biomarker discovery are extremely valuable. In this study, a non-targeted lipidomics strategy using ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was performed to reveal differential lipids between MDD (n = 60) and healthy controls (HCs, n = 60). Validation of changed lipid species was performed in an independent batch including 75 MDD and 52 HC using the same lipidomic method. Pronouncedly changed lipid species in MDD were discovered, which mainly were lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), 1-O-alkyl-2-acyl-PE (PE O), 1-O-alkyl-2-acyl-PC (PC O), sphingomyelin (SM), diacylglycerol (DG), and triacylglycerol (TG). Among these lipid species, LPC, LPE, PC, PE, PI, TG, etc. remarkably increased in MDD and showed pronounced positive relationships with depression severity, while 1-O-alkyl-2-acyl-PE and SM with odd summed carbon number significantly decreased in MDD and demonstrated negative relationships with depression severity. A combinational lipid panel including LPE 20:4, PC 34:1, PI 40:4, SM 39:1, 2, and TG 44:2 was defined as potential diagnostic biomarker with a good sensitivity and specificity for distinguishing MDD from HCs. Our study brings insights into lipid metabolism disorder in MDD and provides a specific potential biomarker for MDD diagnose.

Modelling white-water rafting suitability in a hydropower regulated Alpine River.

PubMed

Carolli, Mauro; Zolezzi, Guido; Geneletti, Davide; Siviglia, Annunziato; Carolli, Fabiano; Cainelli, Oscar

2017-02-01

Cultural and recreational river ecosystem services and their relations with the flow regime are still poorly investigated. We develop a modelling-based approach to assess recreational flow requirements and the spatially distributed river suitability for white-water rafting, a typical service offered by mountain streams, with potential conflicts of interest with hydropower regulation. The approach is based on the principles of habitat suitability modelling using water depth as the main attribute, with preference curves defined through interviews with local rafting guides. The methodology allows to compute streamflow thresholds for conditions of suitability and optimality of a river reach in relation to rafting. Rafting suitability response to past, present and future flow management scenarios can be predicted on the basis of a hydrological model, which is incorporated in the methodology and is able to account for anthropic effects. Rafting suitability is expressed through a novel metric, the "Rafting hydro-suitability index" (RHSI) which quantifies the cumulative duration of suitable and optimal conditions for rafting. The approach is applied on the Noce River (NE Italy), an Alpine River regulated by hydropower production and affected by hydropeaking, which influences suitability at a sub-daily scale. A dedicated algorithm is developed within the hydrological model to resemble hydropeaking conditions with daily flow data. In the Noce River, peak flows associated with hydropeaking support rafting activities in late summer, highlighting the dual nature of hydropeaking in regulated rivers. Rafting suitability is slightly reduced under present, hydropower-regulated flow conditions compared to an idealized flow regime characterised by no water abstractions. Localized water abstractions for small, run-of-the-river hydropower plants are predicted to negatively affect rafting suitability. The proposed methodology can be extended to support decision making for flow

Tunable, Quantitative Fenton-RAFT Polymerization via Metered Reagent Addition.

PubMed

Nothling, Mitchell D; McKenzie, Thomas G; Reyhani, Amin; Qiao, Greg G

2018-05-10

A continuous supply of radical species is a key requirement for activating chain growth and accessing quantitative monomer conversions in reversible addition-fragmentation chain transfer (RAFT) polymerization. In Fenton-RAFT, activation is provided by hydroxyl radicals, whose indiscriminate reactivity and short-lived nature poses a challenge to accessing extended polymerization times and quantitative monomer conversions. Here, an alternative Fenton-RAFT procedure is presented, whereby radical generation can be finely controlled via metered dosing of a component of the Fenton redox reaction (H 2 O 2 ) using an external pumping system. By limiting the instantaneous flux of radicals and ensuring sustained radical generation over tunable time periods, metered reagent addition reduces unwanted radical "wasting" reactions and provides access to consistent quantitative monomer conversions with high chain-end fidelity. Fine tuning of radical concentration during polymerization is achieved simply via adjustment of reagent dose rate, offering significant potential for automation. This modular strategy holds promise for extending traditional RAFT initiation toward more tightly regulated radical concentration profiles and affords excellent prospects for the automation of Fenton-RAFT polymerization. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Formulation and optimisation of raft-forming chewable tablets containing H2 antagonist

PubMed Central

Prajapati, Shailesh T; Mehta, Anant P; Modhia, Ishan P; Patel, Chhagan N

2012-01-01

Purpose: The purpose of this research work was to formulate raft-forming chewable tablets of H2 antagonist (Famotidine) using a raft-forming agent along with an antacid- and gas-generating agent. Materials and Methods: Tablets were prepared by wet granulation and evaluated for raft strength, acid neutralisation capacity, weight variation, % drug content, thickness, hardness, friability and in vitro drug release. Various raft-forming agents were used in preliminary screening. A 23 full-factorial design was used in the present study for optimisation. The amount of sodium alginate, amount of calcium carbonate and amount sodium bicarbonate were selected as independent variables. Raft strength, acid neutralisation capacity and drug release at 30 min were selected as responses. Results: Tablets containing sodium alginate were having maximum raft strength as compared with other raft-forming agents. Acid neutralisation capacity and in vitro drug release of all factorial batches were found to be satisfactory. The F5 batch was optimised based on maximum raft strength and good acid neutralisation capacity. Drug–excipient compatibility study showed no interaction between the drug and excipients. Stability study of the optimised formulation showed that the tablets were stable at accelerated environmental conditions. Conclusion: It was concluded that raft-forming chewable tablets prepared using an optimum amount of sodium alginate, calcium carbonate and sodium bicarbonate could be an efficient dosage form in the treatment of gastro oesophageal reflux disease. PMID:23580933

5-Lipoxygenase contributes to PPARγ activation in macrophages in response to apoptotic cells.

PubMed

von Knethen, Andreas; Sha, Lisa K; Kuchler, Laura; Heeg, Annika K; Fuhrmann, Dominik; Heide, Heinrich; Wittig, Ilka; Maier, Thorsten J; Steinhilber, Dieter; Brüne, Bernhard

2013-12-01

Macrophage polarization to an anti-inflammatory phenotype upon contact with apoptotic cells is a contributing hallmark to immune suppression during the late phase of sepsis. Although the peroxisome proliferator-activated receptor γ (PPARγ) supports this macrophage phenotype switch, it remains elusive how apoptotic cells activate PPARγ. Assuming that a molecule causing PPARγ activation in macrophages originates in the cell membrane of apoptotic cells we analyzed lipid rafts from apoptotic, necrotic, and living human Jurkat T cells which showed the presence of 5-lipoxygenase (5-LO) in lipid rafts of apoptotic cells only. Incubating macrophages with lipid rafts of apoptotic, but not necrotic or living cells, induced PPAR responsive element (PPRE)-driven mRuby reporter gene expression in RAW 264.7 macrophages stably transduced with a 4xPPRE containing vector. Experiments with lipid rafts of apoptotic murine EL4 T cells revealed similar results. To verify the involvement of 5-LO in activating PPARγ in macrophages, Jurkat T cells were incubated with the 5-LO inhibitor MK-866 prior to induction of apoptosis, which failed to induce mRuby expression. Similar results were obtained with lipid rafts of apoptotic EL4 T cells preexposed to the 5-LO inhibitors zileuton and CJ-13610. Interestingly, Jurkat T cells overexpressing 5-LO failed to activate PPARγ in macrophages, while their 5-LO overexpressing apoptotic counterparts did. Our results suggest that during apoptosis 5-LO gets associated with lipid rafts and synthesizes ligands that in turn stimulate PPARγ in macrophages. © 2013.

Effectiveness of LifeRAFT Undergraduate Helping Skills Training Model

ERIC Educational Resources Information Center

Campbell, Elizabeth L.; Davidson, Kenzie; Davidson, Spencer M.

2017-01-01

LifeRAFT, a helping skills training model for undergraduate paraprofessionals, addresses training needs for applied psychology skills for undergraduate psychology majors. LifeRAFT draws from three empirically supported psychotherapy treatments to introduce counselling theory and encourage helping skill progression. Trainees learn practical helping…

Live imaging of prions reveals nascent PrPSc in cell-surface, raft-associated amyloid strings and webs

PubMed Central

Rouvinski, Alexander; Karniely, Sharon; Kounin, Maria; Moussa, Sanaa; Goldberg, Miri D.; Warburg, Gabriela; Lyakhovetsky, Roman; Papy-Garcia, Dulce; Kutzsche, Janine; Korth, Carsten; Carlson, George A.; Godsave, Susan F.; Peters, Peter J.; Luhr, Katarina; Kristensson, Krister

2014-01-01

Mammalian prions refold host glycosylphosphatidylinositol-anchored PrPC into β-sheet–rich PrPSc. PrPSc is rapidly truncated into a C-terminal PrP27-30 core that is stable for days in endolysosomes. The nature of cell-associated prions, their attachment to membranes and rafts, and their subcellular locations are poorly understood; live prion visualization has not previously been achieved. A key obstacle has been the inaccessibility of PrP27-30 epitopes. We overcame this hurdle by focusing on nascent full-length PrPSc rather than on its truncated PrP27-30 product. We show that N-terminal PrPSc epitopes are exposed in their physiological context and visualize, for the first time, PrPSc in living cells. PrPSc resides for hours in unexpected cell-surface, slow moving strings and webs, sheltered from endocytosis. Prion strings observed by light and scanning electron microscopy were thin, micrometer-long structures. They were firmly cell associated, resisted phosphatidylinositol-specific phospholipase C, aligned with raft markers, fluoresced with thioflavin, and were rapidly abolished by anti-prion glycans. Prion strings and webs are the first demonstration of membrane-anchored PrPSc amyloids. PMID:24493590

Raft River Geothermal Area Data Models - Conceptual, Logical and Fact Models

DOE Data Explorer

Cuyler, David

2012-07-19

Conceptual and Logical Data Model for Geothermal Data Concerning Wells, Fields, Power Plants and Related Analyses at Raft River a. Logical Model for Geothermal Data Concerning Wells, Fields, Power Plants and Related Analyses, David Cuyler 2010 b. Fact Model for Geothermal Data Concerning Wells, Fields, Power Plants and Related Analyses, David Cuyler 2010 Derived from Tables, Figures and other Content in Reports from the Raft River Geothermal Project: "Technical Report on the Raft River Geothermal Resource, Cassia County, Idaho," GeothermEx, Inc., August 2002. "Results from the Short-Term Well Testing Program at the Raft River Geothermal Field, Cassia County, Idaho," GeothermEx, Inc., October 2004.

Rapid, Long-Distance Dispersal by Pumice Rafting

PubMed Central

Bryan, Scott E.; Cook, Alex G.; Evans, Jason P.; Hebden, Kerry; Hurrey, Lucy; Colls, Peter; Jell, John S.; Weatherley, Dion; Firn, Jennifer

2012-01-01

Pumice is an extremely effective rafting agent that can dramatically increase the dispersal range of a variety of marine organisms and connect isolated shallow marine and coastal ecosystems. Here we report on a significant recent pumice rafting and long-distance dispersal event that occurred across the southwest Pacific following the 2006 explosive eruption of Home Reef Volcano in Tonga. We have constrained the trajectory, and rate, biomass and biodiversity of transfer, discovering more than 80 species and a substantial biomass underwent a >5000 km journey in 7–8 months. Differing microenvironmental conditions on the pumice, caused by relative stability of clasts at the sea surface, promoted diversity in biotic recruitment. Our findings emphasise pumice rafting as an important process facilitating the distribution of marine life, which have implications for colonisation processes and success, the management of sensitive marine environments, and invasive pest species. PMID:22815770

The role of blood cell membrane lipids on the mode of action of HIV-1 fusion inhibitor sifuvirtide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matos, Pedro M.; Freitas, Teresa; Castanho, Miguel A.R.B.

2010-12-17

Research highlights: {yields} Sifuvirtide interacts with erythrocyte and lymphocyte membrane in a concentration dependent manner by decreasing its dipole potential. {yields} Dipole potential variations in lipid vesicles show sifuvirtide's lipid selectivity towards saturated phosphatidylcholines. {yields} This peptide-membrane interaction may direct the drug towards raft-like membrane domains where the receptors used by HIV are located, facilitating its inhibitory action. -- Abstract: Sifuvirtide is a gp41 based peptide that inhibits HIV-1 fusion with the host cells and is currently under clinical trials. Previous studies showed that sifuvirtide partitions preferably to saturated phosphatidylcholine lipid membranes, instead of fluid-phase lipid vesicles. We extended themore » study to the interaction of the peptide with circulating blood cells, by using the dipole potential sensitive probe di-8-ANEPPS. Sifuvirtide decreased the dipole potential of erythrocyte and lymphocyte membranes in a concentration dependent manner, demonstrating its interaction. Also, the lipid selectivity of the peptide towards more rigid phosphatidylcholines was confirmed based on the dipole potential variations. Overall, the interaction of the peptide with the cell membranes is a contribution of different lipid preferences that presumably directs the peptide towards raft-like domains where the receptors are located, facilitating the reach of the peptide to its molecular target, the gp41 in its pre-fusion conformation.« less

Bioactive Structure of Membrane Lipids and Natural Products Elucidated by a Chemistry-Based Approach.

PubMed

Murata, Michio; Sugiyama, Shigeru; Matsuoka, Shigeru; Matsumori, Nobuaki

2015-08-01

Determining the bioactive structure of membrane lipids is a new concept, which aims to examine the functions of lipids with respect to their three-dimensional structures. As lipids are dynamic by nature, their "structure" does not refer solely to a static picture but also to the local and global motions of the lipid molecules. We consider that interactions with lipids, which are completely defined by their structures, are controlled by the chemical, functional, and conformational matching between lipids and between lipid and protein. In this review, we describe recent advances in understanding the bioactive structures of membrane lipids bound to proteins and related molecules, including some of our recent results. By examining recent works on lipid-raft-related molecules, lipid-protein interactions, and membrane-active natural products, we discuss current perspectives on membrane structural biology. © 2015 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pulse EPR detection of lipid exchange between protein-rich raft and bulk domains in the membrane: methodology development and its application to studies of influenza viral membrane.

PubMed Central

Kawasaki, K; Yin, J J; Subczynski, W K; Hyde, J S; Kusumi, A

2001-01-01

A pulse saturation-recovery electron paramagnetic resonance (EPR) method has been developed that allows estimation of the exchange rates of a spin-labeled lipid between the bulk domain and the protein-rich membrane domain, in which the rate of collision between the spin label and molecular oxygen is reduced (slow-oxygen transport domain, or SLOT domain). It is based on the measurements of saturation-recovery signals of a lipid spin label as a function of concentrations of both molecular oxygen and the spin label. Influenza viral membrane, one of the simplest paradigms for the study of biomembranes, showed the presence of two membrane domains with slow and fast collision rates with oxygen (a 16-fold difference) at 30 degrees C. The outbound rate from and the inbound rate into the SLOT domain (or possibly the rate of the domain disintegration and formation) were estimated to be 7.7 x 10(4) and 4.6 x 10(4) s(-1), (15 micros residency time), respectively, indicating that the SLOT domain is highly dynamic and that the entire SLOT domain represents about one-third of the membrane area. Because the oxygen transport rate in the SLOT domain is a factor of two smaller than that in purple membrane, where bacteriorhodopsin is aggregated, we propose that the SLOT domain in the viral membrane is the cholesterol-rich raft domain stabilized by the trimers of hemagglutinin and/or the tetramers of neuraminidase. PMID:11159441

Prion subcellular fractionation reveals infectivity spectrum, with a high titre-low PrPres level disparity

PubMed Central

2012-01-01

Background Prion disease transmission and pathogenesis are linked to misfolded, typically protease resistant (PrPres) conformers of the normal cellular prion protein (PrPC), with the former posited to be the principal constituent of the infectious 'prion'. Unexplained discrepancies observed between detectable PrPres and infectivity levels exemplify the complexity in deciphering the exact biophysical nature of prions and those host cell factors, if any, which contribute to transmission efficiency. In order to improve our understanding of these important issues, this study utilized a bioassay validated cell culture model of prion infection to investigate discordance between PrPres levels and infectivity titres at a subcellular resolution. Findings Subcellular fractions enriched in lipid rafts or endoplasmic reticulum/mitochondrial marker proteins were equally highly efficient at prion transmission, despite lipid raft fractions containing up to eight times the levels of detectable PrPres. Brain homogenate infectivity was not differentially enhanced by subcellular fraction-specific co-factors, and proteinase K pre-treatment of selected fractions modestly, but equally reduced infectivity. Only lipid raft associated infectivity was enhanced by sonication. Conclusions This study authenticates a subcellular disparity in PrPres and infectivity levels, and eliminates simultaneous divergence of prion strains as the explanation for this phenomenon. On balance, the results align best with the concept that transmission efficiency is influenced more by intrinsic characteristics of the infectious prion, rather than cellular microenvironment conditions or absolute PrPres levels. PMID:22534096

Fire ants self-assemble into waterproof rafts to survive floods

PubMed Central

Mlot, Nathan J.; Tovey, Craig A.; Hu, David L.

2011-01-01

Why does a single fire ant Solenopsis invicta struggle in water, whereas a group can float effortlessly for days? We use time-lapse photography to investigate how fire ants S. invicta link their bodies together to build waterproof rafts. Although water repellency in nature has been previously viewed as a static material property of plant leaves and insect cuticles, we here demonstrate a self-assembled hydrophobic surface. We find that ants can considerably enhance their water repellency by linking their bodies together, a process analogous to the weaving of a waterproof fabric. We present a model for the rate of raft construction based on observations of ant trajectories atop the raft. Central to the construction process is the trapping of ants at the raft edge by their neighbors, suggesting that some “cooperative” behaviors may rely upon coercion. PMID:21518911

Interaction of miltefosine with the lipid and protein components of the erythrocyte membrane.

PubMed

Moreira, Rodrigo Alves; Mendanha, Sebastião Antonio; Hansen, Daiane; Alonso, Antonio

2013-05-01

Miltefosine (MT) is an alkylphospholipid that has been approved for the treatment of breast cancer metastasis and visceral leishmaniasis, although its mechanism of action remains poorly understood. Electron paramagnetic resonance spectroscopy of a spin-labeled lipid and a thiol-specific spin label showed that MT causes an increase in the molecular dynamics of erythrocyte ghost membranes and detergent-resistant membranes (DRMs) prepared from erythrocyte ghosts. In the vesicles of lipid raft constituents, it was shown that 20 mol % sphingomyelin could be replaced by 20 mol % MT with no change in the molecular dynamics. The effect of MT in DRMs was more pronounced than in erythrocyte ghosts, supporting the hypothesis that MT is a lipid raft modulator. At the reported MT-plasma concentrations found during the treatment of leishmaniasis (31-90 µg/mL), our measurements in the blood plasma indicated a hemolytic level of 2%-5%. The experiments indicated that MT acts predominantly on the protein component of the membrane. MT aggregates may wrap around the hydrophobic polypeptide chains, forming micelle-like structures that stabilize protein conformations more exposed to the solvent. Proteins with higher hydrophobicity may induce the penetration of the hydrophilic groups of MT into the membrane and cause it to rupture. Copyright © 2013 Wiley Periodicals, Inc.

Lipid Profiling of the Arabidopsis Hypersensitive Response Reveals Specific Lipid Peroxidation and Fragmentation Processes: Biogenesis of Pimelic and Azelaic Acid1[C][W

PubMed Central

Zoeller, Maria; Stingl, Nadja; Krischke, Markus; Fekete, Agnes; Waller, Frank; Berger, Susanne; Mueller, Martin J.

2012-01-01

Lipid peroxidation (LPO) is induced by a variety of abiotic and biotic stresses. Although LPO is involved in diverse signaling processes, little is known about the oxidation mechanisms and major lipid targets. A systematic lipidomics analysis of LPO in the interaction of Arabidopsis (Arabidopsis thaliana) with Pseudomonas syringae revealed that LPO is predominantly confined to plastid lipids comprising galactolipid and triacylglyceride species and precedes programmed cell death. Singlet oxygen was identified as the major cause of lipid oxidation under basal conditions, while a 13-lipoxygenase (LOX2) and free radical-catalyzed lipid oxidation substantially contribute to the increase upon pathogen infection. Analysis of lox2 mutants revealed that LOX2 is essential for enzymatic membrane peroxidation but not for the pathogen-induced free jasmonate production. Despite massive oxidative modification of plastid lipids, levels of nonoxidized lipids dramatically increased after infection. Pathogen infection also induced an accumulation of fragmented lipids. Analysis of mutants defective in 9-lipoxygenases and LOX2 showed that galactolipid fragmentation is independent of LOXs. We provide strong in vivo evidence for a free radical-catalyzed galactolipid fragmentation mechanism responsible for the formation of the essential biotin precursor pimelic acid as well as of azelaic acid, which was previously postulated to prime the immune response of Arabidopsis. Our results suggest that azelaic acid is a general marker for LPO rather than a general immune signal. The proposed fragmentation mechanism rationalizes the pathogen-induced radical amplification and formation of electrophile signals such as phytoprostanes, malondialdehyde, and hexenal in plastids. PMID:22822212

EGF domain of coagulation factor IX is conducive to exposure of phosphatidylserine.

PubMed

Hidai, Chiaki; Fujiwara, Yusuke; Kokubun, Shinichiro; Kitano, Hisataka

2017-04-01

Lipid rafts are an initiation site for many different signals. Recently, we reported that an EGF domain in activated coagulation factor IX (EGF-F9) increases lipid raft formation and accelerates cell migration. However, the detailed mechanism is not well understood. This study aimed to evaluate the effects of EGF-F9 on the cell membrane. A431 cells (derived from human squamous cell carcinoma) were treated with recombinant EGF-F9. Cells were immunocytochemically stained with probes for lipid rafts or phosphatidylserine (PS). After 3 min of treatment with EGF-F9, cholera toxin subunit B (CTxB) binding domains emerged at the adhesive tips of filopodia. Subsequently, CTxB staining was observed on the filopodial shaft. Finally, large clusters of CTxB domains were observed at the edge of cell bodies. Markers for lipid rafts, such as caveolin-1 and a GPI anchored protein, co-localized with CTxB. Staining with annexin V and XII revealed that PS was exposed at the tips of filopodia, translocated on filopodial shafts, and co-localized with CTxB at the rafts. Immunocytochemistry showed that scramblase-1 protein was present at the filopodial tips. Our data indicates that EGF-F9 accelerates PS exposure around the filopodial adhesion complex and induces clustering of lipid rafts in the cell body. PS exposure is thought to occur on cells undergoing apoptosis. Further study of the function of the EGF-F9 motif in mediating signal transduction is necessary because it is shared by a number of proteins. © 2017 International Federation for Cell Biology.

Inflammation-related alterations of lipids after spinal cord injury revealed by Raman spectroscopy

NASA Astrophysics Data System (ADS)

Tamosaityte, Sandra; Galli, Roberta; Uckermann, Ortrud; Sitoci-Ficici, Kerim H.; Koch, Maria; Later, Robert; Schackert, Gabriele; Koch, Edmund; Steiner, Gerald; Kirsch, Matthias

2016-06-01

Spinal cord injury (SCI) triggers several lipid alterations in nervous tissue. It is characterized by extensive demyelination and the inflammatory response leads to accumulation of activated microglia/macrophages, which often transform into foam cells by accumulation of lipid droplets after engulfment of the damaged myelin sheaths. Using an experimental rat model, Raman microspectroscopy was applied to retrieve the modifications of the lipid distribution following SCI. Coherent anti-Stokes Raman scattering (CARS) and endogenous two-photon fluorescence (TPEF) microscopies were used for the detection of lipid-laden inflammatory cells. The Raman mapping of CH2 deformation mode intensity at 1440 cm-1 retrieved the lipid-depleted injury core. Preserved white matter and inflammatory regions with myelin fragmentation and foam cells were localized by specifically addressing the distribution of esterified lipids, i.e., by mapping the intensity of the carbonyl Raman band at 1743 cm-1, and were in agreement with CARS/TPEF microscopy. Principal component analysis revealed that the inflammatory regions are notably rich in saturated fatty acids. Therefore, Raman spectroscopy enabled to specifically detect inflammation after SCI and myelin degradation products.

On the fate of pumice rafts formed during the 2012 Havre submarine eruption

PubMed Central

Jutzeler, Martin; Marsh, Robert; Carey, Rebecca J.; White, James D. L.; Talling, Peter J.; Karlstrom, Leif

2014-01-01

Pumice rafts are floating mobile accumulations of low-density pumice clasts generated by silicic volcanic eruptions. Pumice in rafts can drift for years, become waterlogged and sink, or become stranded on shorelines. Here we show that the pumice raft formed by the impressive, deep submarine eruption of the Havre caldera volcano (Southwest Pacific) in July 2012 can be mapped by satellite imagery augmented by sailing crew observations. Far from coastal interference, the eruption produced a single >400 km2 raft in 1 day, thus initiating a gigantic, high-precision, natural experiment relevant to both modern and prehistoric oceanic surface dispersal dynamics. Observed raft dispersal can be accurately reproduced by simulating drift and dispersal patterns using currents from an eddy-resolving ocean model hindcast. For future eruptions that produce potentially hazardous pumice rafts, our technique allows real-time forecasts of dispersal routes, in addition to inference of ash/pumice deposit distribution in the deep ocean. PMID:24755668

MPP1 directly interacts with flotillins in erythrocyte membrane - Possible mechanism of raft domain formation.

PubMed

Biernatowska, Agnieszka; Augoff, Katarzyna; Podkalicka, Joanna; Tabaczar, Sabina; Gajdzik-Nowak, Weronika; Czogalla, Aleksander; Sikorski, Aleksander F

2017-11-01

Flotillins are prominent, oligomeric protein components of erythrocyte (RBC) membrane raft domains and are considered to play an important structural role in lateral organization of the plasma membrane. In our previous work on erythroid membranes and giant plasma membrane vesicles (GPMVs) derived from them we have shown that formation of functional domains (resting state rafts) depends on the presence of membrane palmitoylated protein 1 (MPP1/p55), pointing to its new physiological role. Exploration of the molecular mechanism of MPP1 function in organizing membrane domains described here, through searching for its molecular partners in RBC membrane by using different methods, led to the identification of the raft-marker proteins, flotillin 1 and flotillin 2, as hitherto unreported direct MPP1 binding-partners in the RBC membrane. These proteins are found in high molecular-weight complexes in native RBC membrane and, significantly, their presence was shown to be separate from the well-known protein 4.1-dependent interactions of MPP1 with membrane proteins. Furthermore, FLIM analysis revealed that loss of the endogenous MPP1-flotillins interactions resulted in significant changes in RBC membrane-fluidity, emphasizing the physiological importance of such interactions in vivo. Therefore, our data establish a new perspective on the role of MPP1 in erythroid cells and suggests that direct MPP1-flotillins interactions could be the major driving-force behind the formation of raft domains in RBC. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

The foundation mass concrete construction technology of Hongyun Building B tower raft

NASA Astrophysics Data System (ADS)

Liu, Yu; Yin, Suhua; Wu, Yanli; Zhao, Ying

2017-08-01

The foundation of Hongyun building B tower is made of raft board foundation which is 3300mm in the thickness and 2800mm beside side of the core tube. It is researched that the raft foundation mass concrete construction technology is expatiated from temperature and cracks of the raft foundation and the temperature control and monitoring of the concrete base slab construction and concrete curing.

Development of gastroretentive metronidazole floating raft system for targeting Helicobacter pylori.

PubMed

Abou Youssef, Nancy Abdel Hamid; Kassem, Abeer Ahmed; El-Massik, Magda Abd Elsamea; Boraie, Nabila Ahmed

2015-01-01

The study demonstrates the feasibility of prolonging gastric residence time and release rate of metronidazole (Mz) by preparing floating raft system (FRS) using ion-sensitive in situ gel forming polymers. FRSs contained 3, 4, 5 and 0.5, 0.75, 1% w/v sodium alginate (Alg) and gellan gum (G), respectively, 0.25% w/v sodium citrate and calcium carbonate (C). Lipids: glyceryl mono stearate (GMS), Precirol(®) and Compritol(®) were incorporated into G-based formulations (G1%C1%). Mz:lipid ratio was 1:1, except for Mz:GMS, ratios of 1:1.5 and 1:2 were also investigated. Buoyancy, gelation capacity and viscosity parameters were evaluated. Drug release and kinetics for selected formulae were examined. The selected lipid containing formula was subjected to an accelerated stability testing. Alg4%C2% FRS exhibited short gelation lag time (3s), long duration (>24h), floating lag time 1m in and duration >24h, and a reliable sustained drug release (MDT 6h). Gellan gum FRSs achieved successful floating gastroretention, but failed to achieve the required gelation capacity. Incorporation of GMS (Mz:GMS 1:1) enhanced the gelation lag time and duration (6s and >24h, respectively), keeping sustained drug release and formulation stability. The improved characteristics of the selected FRS make them excellent candidates for gastric targeting to eradicate Helicobacter pylori. Copyright © 2015 Elsevier B.V. All rights reserved.

Lipidomics reveals dramatic lipid compositional changes in the maturing postnatal lung

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dautel, Sydney E.; Kyle, Jennifer E.; Clair, Geremy

Lung immaturity is a major cause of morbidity and mortality in premature infants. Understanding the molecular mechanisms driving normal lung development could provide insights on how to ameliorate disrupted development. While transcriptomic and proteomic analyses of normal lung development have been previously reported, characterization of changes in the lipidome is lacking. Lipids play significant roles in the lung, such as dipalmitoylcholine in pulmonary surfactant; however, many of the roles of specific lipid species in normal lung development, as well as in disease states, are not well defined. In this study, we used liquid chromatography-mass spectrometry (LC-MS/MS) to investigate the murinemore » lipidome during normal postnatal lung development. Lipidomics analysis of lungs from post-natal day 7, day 14 and 6-8 week mice (adult) identified 928 unique lipids across 21 lipid subclasses, with dramatic alterations in the lipidome across developmental stages. Our data confirmed previously recognized aspects of post-natal lung development and revealed several insights, including in sphingolipid-mediated apoptosis, inflammation and energy storage/usage. Complementary proteomics, metabolomics and chemical imaging corroborated these observations. Finally, this multi-omic view provides a unique resource and deeper insight into normal pulmonary development.« less

Lipidomics reveals dramatic lipid compositional changes in the maturing postnatal lung

DOE PAGES

Dautel, Sydney E.; Kyle, Jennifer E.; Clair, Geremy; ...

2017-02-01

Lung immaturity is a major cause of morbidity and mortality in premature infants. Understanding the molecular mechanisms driving normal lung development could provide insights on how to ameliorate disrupted development. While transcriptomic and proteomic analyses of normal lung development have been previously reported, characterization of changes in the lipidome is lacking. Lipids play significant roles in the lung, such as dipalmitoylcholine in pulmonary surfactant; however, many of the roles of specific lipid species in normal lung development, as well as in disease states, are not well defined. In this study, we used liquid chromatography-mass spectrometry (LC-MS/MS) to investigate the murinemore » lipidome during normal postnatal lung development. Lipidomics analysis of lungs from post-natal day 7, day 14 and 6-8 week mice (adult) identified 928 unique lipids across 21 lipid subclasses, with dramatic alterations in the lipidome across developmental stages. Our data confirmed previously recognized aspects of post-natal lung development and revealed several insights, including in sphingolipid-mediated apoptosis, inflammation and energy storage/usage. Complementary proteomics, metabolomics and chemical imaging corroborated these observations. Finally, this multi-omic view provides a unique resource and deeper insight into normal pulmonary development.« less

Reduced levels of folate transporters (PCFT and RFC) in membrane lipid rafts result in colonic folate malabsorption in chronic alcoholism.

PubMed

Wani, Nissar Ahmad; Kaur, Jyotdeep

2011-03-01

We studied the effect of chronic ethanol ingestion on folate transport across the colonic apical membranes (CAM) in rats. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20%) solution orally for 3 months and folate transport was studied in the isolated colon apical membrane vesicles. The folate transport was found to be carrier mediated, saturable, with pH optima at 5.0. Chronic ethanol ingestion reduced the folate transport across the CAM by decreasing the affinity of transporters (high Km) for the substrate and by decreasing the number of transporter molecules (low Vmax) on the colon luminal surface. The decreased transport activity at the CAM was associated with down-regulation of the proton-coupled folate transporter (PCFT) and the reduced folate carrier (RFC) which resulted in decreased PCFT and RFC protein levels in the colon of rats fed alcohol chronically. Moreover, the PCFT and the RFC were found to be distributed in detergent insoluble fraction of the CAM in rats. Floatation experiments on Optiprep density gradients demonstrated the association of the PCFT and the RFC protein with lipid rafts (LR). Chronic alcoholism decreased the PCFT and the RFC protein levels in the CAM LR in accordance with the decreased synthesis. Hence, we propose that downregulation in the expression of the PCFT and the RFC in colon results in reduced levels of these transporters in colon apical membrane LR as a mechanism of folate malabsorption during chronic alcoholism. Copyright © 2010 Wiley-Liss, Inc.

Recruitment of mitofusin 2 into “lipid rafts” drives mitochondria fusion induced by Mdivi-1

PubMed Central

Ciarlo, Laura; Vona, Rosa; Manganelli, Valeria; Gambardella, Lucrezia; Raggi, Carla; Marconi, Matteo; Malorni, Walter; Sorice, Maurizio; Garofalo, Tina; Matarrese, Paola

2018-01-01

The regulation of the mitochondrial dynamics and the balance between fusion and fission processes are crucial for the health and fate of the cell. Mitochondrial fusion and fission machinery is controlled by key proteins such as mitofusins, OPA-1 and several further molecules. In the present work we investigated the implication of lipid rafts in mitochondrial fusion induced by Mdivi-1. Our results underscore the possible implication of lipid “rafts” in mitochondrial morphogenetic changes and their homeostasis. PMID:29721168

Endoplasmic reticulum factor ERLIN2 regulates cytosolic lipid content in cancer cells

PubMed Central

Wang, Guohui; Zhang, Xuebao; Lee, Jin-Sook; Wang, Xiaogang; Yang, Zeng-Quan; Zhang, Kezhong

2013-01-01

Increased de novo lipogenesis is a hallmark of aggressive cancers. Lipid droplets, the major form of cytosolic lipid storage, have been implicated in cancer cell proliferation and tumorigenesis. Recently, we identified the ERLIN2 [ER (endoplasmic reticulum) lipid raft-associated 2) gene that is amplified and overexpressed in aggressive human breast cancer. Previous studies demonstrated that ERLIN2 plays a supporting oncogenic role by facilitating the transformation of human breast cancer cells. In the present study, we found that ERLIN2 supports cancer cell growth by regulating cytosolic lipid droplet production. ERLIN2 is preferably expressed in human breast cancer cells or hepatoma cells and is inducible by insulin signalling or when cells are cultured in lipoprotein-deficient medium. Increased expression of ERLIN2 promotes the accumulation of cytosolic lipid droplets in breast cancer cells or hepatoma cells in response to insulin or overload of unsaturated fatty acids. ERLIN2 regulates activation of SREBP (sterol regulatory element-binding protein) 1c, the key regulator of de novo lipogenesis, in cancer cells. ERLIN2 was found to bind to INSIG1 (insulin-induced gene 1), a key ER membrane protein that blocks SREBP activation. Consistent with the role of ERLIN2 in regulating cytosolic lipid content, down-regulation of ERLIN2 in breast cancer or hepatoma cells led to lower cell proliferation rates. The present study revealed a novel role for ERLIN2 in supporting cancer cell growth by promoting the activation of the key lipogenic regulator SREBP1c and the production of cytosolic lipid droplets. The identification of ERLIN2 as a regulator of cytosolic lipid content in cancer cells has important implications for understanding the molecular basis of tumorigenesis and the treatment of cancer. PMID:22690709

Improvement of Aconitum napellus micropropagation by liquid culture on floating membrane rafts.

PubMed

Watad, A A; Kochba, M; Nissim, A; Gaba, V

1995-03-01

An efficient method was developed using floating membrane rafts (Liferaft(™)) for the micropropagation of Aconitum napellus (Ranunculaceae), a cut flower crop with a low natural propagation rate. This was achieved by introducing shoot tips into culture on Murashige and Skoog's (1962) solid medium, or liquid medium-supported rafts, supplemented by different levels of benzyl adenine (BA). Optimum shoot proliferation on solid medium required 4mg/l BA, whereas for expiants supported on rafts optimal proliferation was achieved at 0.25mg/l BA. Maximum shoot proliferation was found using the floating rafts (propagation ratio of 4.2 per month), 45% higher than the maximum value on solid medium. A similar value could be obtained on solid medium after a period of 2 months. The optimal response to BA was similar for fresh weight gain and shoot length. Growth in a shallow layer of liquid in shake flasks gives a similar shoot multiplication rate to that on floating rafts; however, submerged leaves brown and die.

Self-Assembled Core-Shell-Type Lipid-Polymer Hybrid Nanoparticles: Intracellular Trafficking and Relevance for Oral Absorption.

PubMed

Li, Qiuxia; Xia, Dengning; Tao, Jinsong; Shen, Aijun; He, Yuan; Gan, Yong; Wang, Chi

2017-10-01

Lipid-polymer hybrid nanoparticles (NPs) are advantageous for drug delivery. However, their intracellular trafficking mechanism and relevance for oral drug absorption are poorly understood. In this study, self-assembled core-shell lipid-polymer hybrid NPs made of poly(lactic-co-glycolic acid) (PLGA) and various lipids were developed to study their differing intracellular trafficking in intestinal epithelial cells and their relevance for oral absorption of a model drug saquinavir (SQV). Our results demonstrated that the endocytosis and exocytosis of hybrid NPs could be changed by varying the kind of lipid. A glyceride mixture (hybrid NPs-1) decreased endocytosis but increased exocytosis in Caco-2 cells, whereas the phospholipid (E200) (hybrid NPs-2) decreased endocytosis but exocytosis was unaffected as compared with PLGA nanoparticles. The transport of hybrid NPs-1 in cells involved various pathways, including caveolae/lipid raft-dependent endocytosis, and clathrin-mediated endocytosis and macropinocytosis, which was different from the other groups of NPs that involved only caveolae/lipid raft-dependent endocytosis. Compared with that of the reference formulation (nanoemulsion), the oral absorption of SQV-loaded hybrid NPs in rats was poor, probably due to the limited drug release and transcytosis of NPs across the intestinal epithelium. In conclusion, the intracellular processing of hybrid NPs in intestinal epithelia can be altered by adding lipids to the NP. However, it appears unfavorable to use PLGA-based NPs to improve oral absorption of SQV compared with nanoemulsion. Our findings will be essential in the development of polymer-based NPs for the oral delivery of drugs with the purpose of improving their oral absorption. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Organochlorine Pesticides (OCPs) in raft mussels coming from Galician rías.

PubMed

Carro, Nieves; García, Isabel; Ignacio, María; Mouteira, Ana

2017-09-01

This survey gives an overview of the range of organochlorine pesticides (OCPs) α and γ-HCH (lindane), HCB, pp´-DDE, pp´-DDD, op´-DDT, pp´-DDT, aldrin, dieldrin, endrin, isodrin and trans-nonachlor in raft mussels cultivated in several polygons of the Galician Rías (Ares-Betanzos, Muros-Noia, Arousa, Pontevedra and Vigo) during the period 1998-2014. A significant number of OCPs were detected, but the levels found were generally low, mainly in the recent samplings (2010-2014). The levels of ∑DDs (sum of DDTs and its metabolites, pp´-DDE and pp´-DDD) ranged between 0.22 ng/g dry weight (dw) in samples from the Ría of Pontevedra and 46.4 ng/g dw in mussels from the Ría of Arousa. Both aldrin and endrin were not detected or were below the analytical limits of detection. With regard to other pesticides, dieldrin is the major compound, followed by γ-HCH, isodrin, α-HCH and transnonachlor. Significant ANOVA regressions indicated variations in levels of some OCPs with the biological parameters (lipid, condition index and shell length). Univariate and multivariate analysis of OCP levels revealed no significant differences between Rías. Temporal trends showed an important decline of OCPs in the studied samples during the period 1998-2014.

Association of Membrane Rafts and Postsynaptic Density: Proteomics, Biochemical, and Ultrastructural Analyses

PubMed Central

Suzuki, Tatsuo; Zhang, Jingping; Miyazawa, Shoko; Liu, Qian; Farzan, Michael R.; Yao, Wei-Dong

2011-01-01

Postsynaptic membrane rafts are believed to play important roles in synaptic signaling, plasticity, and maintenance. However, their molecular identities remain elusive. Further, how they interact with the well-established signaling specialization, the postsynaptic density (PSD), is poorly understood. We previously detected a number of conventional PSD proteins in detergent-resistant membranes (DRMs). Here, we have performed LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) analyses on postsynaptic membrane rafts and PSDs. Our comparative analysis identified an extensive overlap of protein components in the two structures. This overlapping could be explained, at least partly, by a physical association of the two structures. Meanwhile, a significant number of proteins displayed biased distributions to either rafts or PSDs, suggesting distinct roles for the two postsynaptic specializations. Using biochemical and electron microscopic methods, we directly detected membrane raft-PSD complexes. In vitro reconstitution experiments indicated that the formation of raft-PSD complexes was not due to the artificial reconstruction of once-solubilized membrane components and PSD structures, supporting that these complexes occurred in vivo. Taking together, our results provide evidence that postsynaptic membrane rafts and PSDs may be physically associated. Such association could be important in postsynaptic signal integration, synaptic function, and maintenance. PMID:21797867

Raptor ecology of Raft River Valley, Idaho

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thurow, T.L.; White, C.M.; Howard, R.P.

1980-09-01

Raptor data were gathered in the 988-km/sup 2/ Raft River Valley in southcentral Idaho while conducting a tolerance study on the nesting Ferruginous Hawk (Buteo regalis) near the Department of Energy's Raft River Geothermal Site. Prior research from 1972 to 1977 on the nesting activity of the Ferruginous Hawk population provided a historical information base. These data are combined with new Ferruginous Hawk data collected between 1978 and 1980 to give a continuous 9-year breeding survey. Information on the distribution, density, and production of the other raptor species found in the study area during 1978 and 1979 is also provided.

Study on design method and vibration reduction characteristic of floating raft with periodic structure

NASA Astrophysics Data System (ADS)

Fang, Yuanyuan; Zuo, Yanyan; Xia, Zhaowang

2018-03-01

The noise level is getting higher with the development of high-power marine power plant. Mechanical noise is one of the most obvious noise sources which not only affect equipment reliability, riding comfort and working environment, but also enlarge underwater noise. The periodic truss type device which is commonly applied in fields of aerospace and architectural is introduced to floating raft construction in ship. Four different raft frame structure are designed in the paper. The vibration transmissibility is taken as an evaluation index to measure vibration isolation effect. A design scheme with the best vibration isolation effect is found by numerical method. Plate type and the optimized periodic truss type raft frame structure are processed to experimental verify vibration isolation effect of the structure of the periodic raft. The experimental results demonstrate that the same quality of the periodic truss floating raft has better isolation effect than that of the plate type floating raft.

Reversible Addition Fragmentation Chain Transfer (RAFT) Polymerization of 4-Vinylbenzaldehyde

PubMed Central

Sun, Guorong; Cheng, Chong; Wooley, Karen L.

2008-01-01

The direct reversible addition fragmentation chain transfer (RAFT) polymerization of 4-vinylbenzaldehyde (VBA) was established as a new synthetic method for the preparation of well-defined poly(vinylbenzaldehyde) (PVBA), a polymer having reactive aldehyde side chain substiuents. RAFT polymerization of VBA was investigated using S-1-dodecyl-S’-(α,α’-dimethyl-α”-acetic acid)trithiocarbonate (DDMAT) as chain transfer agent (CTA) and 2,2′-azobis(isobutyronitrile) (AIBN) as initiator in 1,4-dioxane or 2-butanone at 70-75 °C for 7.5-22.5 h. With 45-76% of monomer conversion, the resulting PVBA had well controlled number-average molecular weight (Mn) and low polydispersity (PDI < 1.17). The living characteristic of the RAFT polymerization process was confirmed by the linearity between the Mn values of PVBA and monomer conversions. Well-defined PVBA was further used as a macromolecular chain transfer agent (macro-CTA) in RAFT polymerization of styrene (St), and a block copolymer PVBA-b-PSt with relatively low polydispersity (PDI = 1.20) was successfully synthesized. PMID:19066633

Comprehensive Lipidome-Wide Profiling Reveals Dynamic Changes of Tea Lipids during Manufacturing Process of Black Tea.

PubMed

Li, Jia; Hua, Jinjie; Zhou, Qinghua; Dong, Chunwang; Wang, Jinjin; Deng, Yuliang; Yuan, Haibo; Jiang, Yongwen

2017-11-22

As important biomolecules in Camellia sinensis L., lipids undergo substantial changes during black tea manufacture, which is considered to contribute to tea sensory quality. However, limited by analytical capacity, detailed lipid composition and its dynamic changes during black tea manufacture remain unclear. Herein, we performed tea lipidome profiling using high resolution liquid chromatography coupled to mass spectrometry (LC-MS), which allows simultaneous and robust analysis of 192 individual lipid species in black tea, covering 17 (sub)classes. Furthermore, dynamic changes of tea lipids during black tea manufacture were investigated. Significant alterations of lipid pattern were revealed, involved with chlorophyll degradation, metabolic pathways of glycoglycerolipids, and other extraplastidial membrane lipids. To our knowledge, this report presented most comprehensive coverage of lipid species in black tea. This study provides a global and in-depth metabolic map of tea lipidome during black tea manufacture.

Stabilization of composition fluctuations in mixed membranes by hybrid lipids

NASA Astrophysics Data System (ADS)

Safran, Samuel; Palmieri, Benoit

2013-03-01

A ternary mixture model is proposed to describe composition fluctuations in mixed membranes composed of saturated, unsaturated and hybrid lipids. The asymmetric hybrid lipid has one saturated and one unsaturated hydrocarbon chain and it can reduce the packing incompatibility between saturated and unsaturated lipids. A methodology to recast the free-energy of the lattice in terms of a continuous isotropic field theory is proposed and used to analyze composition fluctuations above the critical temperature. The effect of hybrid lipids on fluctuations domains rich in saturated/unsaturated lipids is predicted. The correlation length of such fluctuations decreases significantly with increasing amounts of hybrids even if the temperature is maintained close to the critical temperature. This provides an upper bound for the domain sizes expected in rafts stabilized by hybrids, above the critical temperature. When the hybrid composition of the membrane is increased further, a crossover value is found above which ``stripe-like'' fluctuations are observed. The wavelength of these fluctuations decreases with increasing hybrid fraction and tends toward a molecular size in a membrane that contains only hybrids.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yi, Jae-Sung; Choo, Hyo-Jung; Cho, Bong-Rae

Lipid rafts are plasma membrane platforms mediating signal transduction pathways for cellular proliferation, differentiation and apoptosis. Here, we show that membrane fluidity was increased in HeLa cells following treatment with ginsenoside Rh2 (Rh2), as determined by cell staining with carboxy-laurdan (C-laurdan), a two-photon dye designed for measuring membrane hydrophobicity. In the presence of Rh2, caveolin-1 appeared in non-raft fractions after sucrose gradient ultracentrifugation. In addition, caveolin-1 and GM1, lipid raft landmarkers, were internalized within cells after exposure to Rh2, indicating that Rh2 might disrupt lipid rafts. Since cholesterol overloading, which fortifies lipid rafts, prevented an increase in Rh2-induced membrane fluidity,more » caveolin-1 internalization and apoptosis, lipid rafts appear to be essential for Rh2-induced apoptosis. Moreover, Rh2-induced Fas oligomerization was abolished following cholesterol overloading, and Rh2-induced apoptosis was inhibited following treatment with siRNA for Fas. This result suggests that Rh2 is a novel lipid raft disruptor leading to Fas oligomerization and apoptosis.« less

Regulation of Lipid Metabolism by Dicer Revealed through SILAC Mice

PubMed Central

Huang, Tai-Chung; Saharabuddhe, Nandini A.; Kim, Min-Sik; Getnet, Derese; Yang, Yi; Peterson, Jonathan M.; Ghosh, Bidyut; Chaerkady, Raghothama; Leach, Steven D.; Marchionni, Luigi; Wong, G. William; Pandey, Akhilesh

2012-01-01

Dicer is a ribonuclease whose major role is to generate mature microRNAs although additional functions have been proposed. Deletion of Dicer leads to embryonic lethality in mice. To study the role of Dicer in adults, we generated mice in which administration of tamoxifen induces deletion of Dicer. Surprisingly, disruption of Dicer in adult mice induced lipid accumulation in the small intestine. To dissect the underlying mechanisms, we carried out miRNA, mRNA and proteomic profiling of small intestine. The proteomic analysis was done using mice metabolically labeled with heavy lysine (SILAC mice) for an in vivo readout. We identified 646 proteins of which 80 were upregulated >2-fold and 75 were downregulated. Consistent with the accumulation of lipids, Dicer disruption caused a marked decrease of microsomal triglyceride transfer protein, long-chain fatty acyl-CoA ligase 5, fatty acid binding protein, and very-long-chain fatty acyl-CoA dehydrogenase, among others. We validated these results using multiple reaction monitoring (MRM) experiments by targeting proteotypic peptides. Our data reveal a previously unappreciated role of Dicer in lipid metabolism. These studies demonstrate a systems biology approach by integrating mouse models, metabolic labeling, gene expression profiling and quantitative proteomics can be a powerful tool for understanding complex biological systems. PMID:22313051

33 CFR 100.102 - Great Connecticut River Raft Race, Middletown, CT.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Great Connecticut River Raft Race... Raft Race, Middletown, CT. (a) Regulated Area. That section of the Connecticut River between Dart.... (1) The Coast Guard patrol commander may delay, modify, or cancel the race as conditions or...

Influence of coarsened and rafted microstructures on the thermomechanical fatigue of a Ni-base superalloy

DOE PAGES

Kirka, M. M.; Brindley, K. A.; Neu, R. W.; ...

2015-08-17

The aging of the microstructure of Ni-base superalloys during service is mainly characterized by coarsening and rafting of the γ' precipitates. The influence of these different aged microstructures on thermomechanical fatigue (TMF) under either continuously cycled (CC) and creep-fatigue (CF) was investigated. Three different aged microstructures, generated through accelerated aging and pre-creep treatments, were studied: stress-free coarsened γ', rafted with orientation perpendicular to loading direction (N-raft), and rafted with orientation parallel to loading direction (P-raft). Under most conditions, the aged microstructures were less resistant to TMF than the virgin microstructure; however, there were exceptions. Both stress-free coarsened and N-raft microstructuresmore » resulted in a reduction in TMF life under both CC and CF conditions in comparison to the virgin material. P-raft microstructure also resulted in reduction in TMF life under CC conditions; however, an increase in life over that of the virgin material was observed under CF conditions. Finally, these differences are discussed and hypothesized to be related to the interactions of the dislocations in the γ channels with γ' precipitates.« less

Canine epidermal lipid sampling by skin scrub revealed variations between different body sites and normal and atopic dogs

PubMed Central

2014-01-01

Background Previously, we evaluated a minimally invasive epidermal lipid sampling method called skin scrub, which achieved reproducible and comparable results to skin scraping. The present study aimed at investigating regional variations in canine epidermal lipid composition using the skin scrub technique and its suitability for collecting skin lipids in dogs suffering from certain skin diseases. Eight different body sites (5 highly and 3 lowly predisposed for atopic lesions) were sampled by skin scrub in 8 control dogs with normal skin. Additionally, lesional and non-lesional skin was sampled from 12 atopic dogs and 4 dogs with other skin diseases by skin scrub. Lipid fractions were separated by high performance thin layer chromatography and analysed densitometrically. Results No significant differences in total lipid content were found among the body sites tested in the control dogs. However, the pinna, lip and caudal back contained significantly lower concentrations of ceramides, whereas the palmar metacarpus and the axillary region contained significantly higher amounts of ceramides and cholesterol than most other body sites. The amount of total lipids and ceramides including all ceramide classes were significantly lower in both lesional and non-lesional skin of atopic dogs compared to normal skin, with the reduction being more pronounced in lesional skin. The sampling by skin scrub was relatively painless and caused only slight erythema at the sampled areas but no oedema. Histological examinations of skin biopsies at 2 skin scrubbed areas revealed a potential lipid extraction from the transition zone between stratum corneum and granulosum. Conclusions The present study revealed regional variations in the epidermal lipid and ceramide composition in dogs without skin abnormalities but no connection between lipid composition and predilection sites for canine atopic dermatitis lesions. The skin scrub technique proved to be a practicable sampling method for canine

Castaways can't be choosers - Homogenization of rafting assemblages on floating seaweeds

NASA Astrophysics Data System (ADS)

Gutow, Lars; Beermann, Jan; Buschbaum, Christian; Rivadeneira, Marcelo M.; Thiel, Martin

2015-01-01

After detachment from benthic habitats, the epibiont assemblages on floating seaweeds undergo substantial changes, but little is known regarding whether succession varies among different seaweed species. Given that floating algae may represent a limiting habitat in many regions, rafting organisms may be unselective and colonize any available seaweed patch at the sea surface. This process may homogenize rafting assemblages on different seaweed species, which our study examined by comparing the assemblages on benthic and floating individuals of the fucoid seaweeds Fucus vesiculosus and Sargassum muticum in the northern Wadden Sea (North Sea). Species richness was about twice as high on S. muticum as on F. vesiculosus, both on benthic and floating individuals. In both seaweed species benthic samples were more diverse than floating samples. However, the species composition differed significantly only between benthic thalli, but not between floating thalli of the two seaweed species. Separate analyses of sessile and mobile epibionts showed that the homogenization of rafting assemblages was mainly caused by mobile species. Among these, grazing isopods from the genus Idotea reached extraordinarily high densities on the floating samples from the northern Wadden Sea, suggesting that the availability of seaweed rafts was indeed limiting. Enhanced break-up of algal rafts associated with intense feeding by abundant herbivores might force rafters to recolonize benthic habitats. These colonization processes may enhance successful dispersal of rafting organisms and thereby contribute to population connectivity between sink populations in the Wadden Sea and source populations from up-current regions.

Comperative studies with Culex pipiens egg rafts. Immunogenetic, electrophoretic and enzymatic analysis of unfertilized, compatible and incompatible fertilized eggs.

PubMed

Schumann, W

1974-01-01

By applying immunologic, electrophoretic and enzymatic methods, extracts of different raft types of Culex pipiens were analysed. Rafts of the crosses Pa x Pa and Ha x Ha contained four common antigens, while unfertilized rafts of Pa and Ha (no antisera were prepared against them) and rafts of the crosses Og x Og, Og x Pa, and Pa x Og shared three common antigens with the remaining raft extracts. Disk-electrophoresis of raft extracts in acrylamide gel resulted in different electropherograms. Ten protein bands were common to all these raft types. The unfertilized rafts of Pa and Ha yielded three more protein bands, the crosses Pa x Ha and Ha x Pa one more, the crosses Og x Og and Pa x Og three more, and Og x Pa two more. Many enzymes were demonstrated in the raft extracts after they were separated in acrylamide gel and incubated with the corresponding substrate solutions. All the raft types possessed one enzyme type for glutaminate-, lactate-, glucose-6-phosphate-dehydrogenase and catalase. Malate-dehydrogenase and leucine aminopeptidase occurred in each raft type as two isoenzymes. Alkaline phosphatase was observed as a single enzyme, but was lacking in rafts of the crosses Pa X Pa and Ha X Ha. While rafts of the crosses Og x Og and Og x Pa possessed two acid phosphatases, three could be demonstrated for the remaining raft types. Up to eight esterases appeared; rafts of the crosses Og x Og and Og x Pa possessed seven such activities. The results obtained by the Ouchterlony test, disk-electrophoresis and the histochemical enzyme tests are discussed in context and checked according to the phenomenon of incompatibility.

Poly(amidoamine) dendrimers on lipid bilayers II: Effects of bilayer phase and dendrimer termination.

PubMed

Kelly, Christopher V; Leroueil, Pascale R; Orr, Bradford G; Banaszak Holl, Mark M; Andricioaei, Ioan

2008-08-07

The molecular structures and enthalpy release of poly(amidoamine) (PAMAM) dendrimers binding to 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) bilayers were explored through atomistic molecular dynamics. Three PAMAM dendrimer terminations were examined: protonated primary amine, neutral acetamide, and deprotonated carboxylic acid. Fluid and gel lipid phases were examined to extract the effects of lipid tail mobility on the binding of generation-3 dendrimers, which are directly relevant to the nanoparticle interactions involving lipid rafts, endocytosis, lipid removal, and/or membrane pores. Upon binding to gel phase lipids, dendrimers remained spherical, had a constant radius of gyration, and approximately one-quarter of the terminal groups were in close proximity to the lipids. In contrast, upon binding to fluid phase bilayers, dendrimers flattened out with a large increase in their asphericity and radii of gyration. Although over twice as many dendrimer-lipid contacts were formed on fluid versus gel phase lipids, the dendrimer-lipid interaction energy was only 20% stronger. The greatest enthalpy release upon binding was between the charged dendrimers and the lipid bilayer. However, the stronger binding to fluid versus gel phase lipids was driven by the hydrophobic interactions between the inner dendrimer and lipid tails.

The Effects of Polyunsaturated Lipid Components on bilayer Structure

NASA Astrophysics Data System (ADS)

Pramudya, Y.; Kiss, A.; Nguyen, Lam T.; Yuan, J.; Hirst, Linda S.

2007-03-01

Polyunsaturated fatty acids (PUFAs), such as DHA (Docosahexanoic Acid) and AA (Alphalinoleic Acid) have been the focus of much research attention in recent years, due to their apparent health benefits and effects on cell physiology. They are found in a variety of biological membranes and have been implicated with lipid raft formation and possible function, particularly in the retinal rod cells and the central nervous system. In this work lipid bilayer structure has been investigated in lipid mixtures, incorporating polyunsaturated fatty acid moieties. The structural effects of increasing concentrations of both symmetric and asymmetric PUFA materials on the bilayer structure are investigated via synchrotron x-ray diffraction on solution samples. We observe bilayer spacings to increase with the percentage of unsaturated fatty acid lipid in the membrane, whilst the degree of ordering significantly decreases. In fact above 20% of fatty acid, well defined bilayers are no longer observed to form. Evidence of phase separation can be clearly seen from these x-ray results and in combination with AFM measurements.

DHA-fluorescent probe is sensitive to membrane order and reveals molecular adaptation of DHA in ordered lipid microdomains☆

PubMed Central

Teague, Heather; Ross, Ron; Harris, Mitchel; Mitchell, Drake C.; Shaikh, Saame Raza

2012-01-01

Docosahexaenoic acid (DHA) disrupts the size and order of plasma membrane lipid microdomains in vitro and in vivo. However, it is unknown how the highly disordered structure of DHA mechanistically adapts to increase the order of tightly packed lipid microdomains. Therefore, we studied a novel DHA-Bodipy fluorescent probe to address this issue. We first determined if the DHA-Bodipy probe localized to the plasma membrane of primary B and immortal EL4 cells. Image analysis revealed that DHA-Bodipy localized into the plasma membrane of primary B cells more efficiently than EL4 cells. We then determined if the probe detected changes in plasma membrane order. Quantitative analysis of time-lapse movies established that DHA-Bodipy was sensitive to membrane molecular order. This allowed us to investigate how DHA-Bodipy physically adapted to ordered lipid microdomains. To accomplish this, we employed steady-state and time-resolved fluorescence anisotropy measurements in lipid vesicles of varying composition. Similar to cell culture studies, the probe was highly sensitive to membrane order in lipid vesicles. Moreover, these experiments revealed, relative to controls, that upon incorporation into highly ordered microdomains, DHA-Bodipy underwent an increase in its fluorescence lifetime and molecular order. In addition, the probe displayed a significant reduction in its rotational diffusion compared to controls. Altogether, DHA-Bodipy was highly sensitive to membrane order and revealed for the first time that DHA, despite its flexibility, could become ordered with less rotational motion inside ordered lipid microdomains. Mechanistically, this explains how DHA acyl chains can increase order upon formation of lipid microdomains in vivo. PMID:22841541