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Sample records for lipid rafts reveals

  1. Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors

    PubMed Central

    Foster, Leonard J.; de Hoog, Carmen L.; Mann, Matthias

    2003-01-01

    Membrane lipids were once thought to be homogenously distributed in the 2D surface of a membrane, but the lipid raft theory suggests that cholesterol and sphingolipids partition away from other membrane lipids. Lipid raft theory further implicates these cholesterol-rich domains in many processes such as signaling and vesicle traffic. However, direct characterization of rafts has been difficult, because they cannot be isolated in pure form. In the first functional proteomic analysis of rafts, we use quantitative high-resolution MS to specifically detect proteins depleted from rafts by cholesterol-disrupting drugs, resulting in a set of 241 authentic lipid raft components. We detect a large proportion of signaling molecules, highly enriched versus total membranes and detergent-resistant fractions, which thus far biochemically defined rafts. Our results provide the first large-scale and unbiased evidence, to our knowledge, for the connection of rafts with signaling and place limits on the fraction of plasma membrane composed by rafts. PMID:12724530

  2. RaftProt: mammalian lipid raft proteome database

    PubMed Central

    Shah, Anup; Chen, David; Boda, Akash R.; Foster, Leonard J.; Davis, Melissa J.; Hill, Michelle M.

    2015-01-01

    RaftProt (http://lipid-raft-database.di.uq.edu.au/) is a database of mammalian lipid raft-associated proteins as reported in high-throughput mass spectrometry studies. Lipid rafts are specialized membrane microdomains enriched in cholesterol and sphingolipids thought to act as dynamic signalling and sorting platforms. Given their fundamental roles in cellular regulation, there is a plethora of information on the size, composition and regulation of these membrane microdomains, including a large number of proteomics studies. To facilitate the mining and analysis of published lipid raft proteomics studies, we have developed a searchable database RaftProt. In addition to browsing the studies, performing basic queries by protein and gene names, searching experiments by cell, tissue and organisms; we have implemented several advanced features to facilitate data mining. To address the issue of potential bias due to biochemical preparation procedures used, we have captured the lipid raft preparation methods and implemented advanced search option for methodology and sample treatment conditions, such as cholesterol depletion. Furthermore, we have identified a list of high confidence proteins, and enabled searching only from this list of likely bona fide lipid raft proteins. Given the apparent biological importance of lipid raft and their associated proteins, this database would constitute a key resource for the scientific community. PMID:25392410

  3. 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…

  4. Lipid Raft in Cardiac Health and Disease

    PubMed Central

    Das, Manika; Das, Dipak K

    2009-01-01

    Lipid rafts are sphingolipid and cholesterol rich micro-domains of the plasma membrane that coordinate and regulate varieties of signaling processes. Lipid rafts are also present in cardiac myocytes and are enriched in signaling molecules and ion channel regulatory proteins. Lipid rafts are receiving increasing attention as cellular organelles contributing to the pathogenesis of several structural and functional processes including cardiac hypertrophy and heart failure. At present, very little is known about the role of lipid rafts in cardiac function and dysfunction. This review will discuss the possible role of lipid rafts in cardiac health and disease. PMID:20436850

  5. Lipid rafts: heterogeneity on the high seas.

    PubMed Central

    Pike, Linda J

    2004-01-01

    Lipid rafts are membrane microdomains that are enriched in cholesterol and glycosphingolipids. They have been implicated in processes as diverse as signal transduction, endocytosis and cholesterol trafficking. Recent evidence suggests that this diversity of function is accompanied by a diversity in the composition of lipid rafts. The rafts in cells appear to be heterogeneous both in terms of their protein and their lipid content, and can be localized to different regions of the cell. This review summarizes the data supporting the concept of heterogeneity among lipid rafts and outlines the evidence for cross-talk between raft components. Based on differences in the ways in which proteins interact with rafts, the Induced-Fit Model of Raft Heterogeneity is proposed to explain the establishment and maintenance of heterogeneity within raft populations. PMID:14662007

  6. Exploring the Existence of Lipid Rafts in Bacteria

    PubMed Central

    2015-01-01

    SUMMARY An interesting concept in the organization of cellular membranes is the proposed existence of lipid rafts. Membranes of eukaryotic cells organize signal transduction proteins into membrane rafts or lipid rafts that are enriched in particular lipids such as cholesterol and are important for the correct functionality of diverse cellular processes. The assembly of lipid rafts in eukaryotes has been considered a fundamental step during the evolution of cellular complexity, suggesting that bacteria and archaea were organisms too simple to require such a sophisticated organization of their cellular membranes. However, it was recently discovered that bacteria organize many signal transduction, protein secretion, and transport processes in functional membrane microdomains, which are equivalent to the lipid rafts of eukaryotic cells. This review contains the most significant advances during the last 4 years in understanding the structural and biological role of lipid rafts in bacteria. Furthermore, this review shows a detailed description of a number of molecular and genetic approaches related to the discovery of bacterial lipid rafts as well as an overview of the group of tentative lipid-protein and protein-protein interactions that give consistency to these sophisticated signaling platforms. Additional data suggesting that lipid rafts are widely distributed in bacteria are presented in this review. Therefore, we discuss the available techniques and optimized protocols for the purification and analysis of raft-associated proteins in various bacterial species to aid in the study of bacterial lipid rafts in other laboratories that could be interested in this topic. Overall, the discovery of lipid rafts in bacteria reveals a new level of sophistication in signal transduction and membrane organization that was unexpected for bacteria and shows that bacteria are more complex than previously appreciated. PMID:25652542

  7. Native low density lipoprotein promotes lipid raft formation in macrophages

    PubMed Central

    SONG, JIAN; PING, LING-YAN; DUONG, DUC M.; GAO, XIAO-YAN; HE, CHUN-YAN; WEI, LEI; WU, JUN-ZHU

    2016-01-01

    Oxidized low-density lipoprotein (LDL) has an important role in atherogenesis; however, the mechanisms underlying cell-mediated LDL oxidation remain to be elucidated. The present study investigated whether native-LDL induced lipid raft formation, in order to gain further insight into LDL oxidation. Confocal microscopic analysis revealed that lipid rafts were aggregated or clustered in the membrane, which were colocalized with myeloperoxidase (MPO) upon native LDL stimulation; however, in the presence of methyl-β-cyclodextrin (MβCD), LDL-stimulated aggregation, translocation, and colocalization of lipid rafts components was abolished.. In addition, lipid raft disruptors MβCD and filipin decreased malondialdehyde expression levels. Density gradient centrifugation coupled to label-free quantitative proteomic analysis identified 1,449 individual proteins, of which 203 were significantly upregulated following native-LDL stimulation. Functional classification of the proteins identified in the lipid rafts revealed that the expression levels of translocation proteins were upregulated. In conclusion, the results of the present study indicated that native-LDL induced lipid raft clustering in macrophages, and the expression levels of several proteins were altered in the stimulated macrophages, which provided novel insights into the mechanism underlying LDL oxidation. PMID:26781977

  8. Lipid Raft: A Floating Island Of Death or Survival

    PubMed Central

    George, Kimberly S.; Wu, Shiyong

    2012-01-01

    Lipid rafts are microdomains of the plasma membrane enriched in cholesterol and sphingolipids, and play an important role in the initiation of many pharmacological agent-induced signaling pathways and toxicological effects. The structure of lipid rafts is dynamic, resulting in an ever-changing content of both lipids and proteins. Cholesterol, as a major component of lipid rafts, is critical for the formation and configuration of lipid rafts microdomains, which provide signaling platforms capable of activating both pro-apoptotic and anti-apoptotic signaling pathways. A change of cholesterol level can result in lipid rafts disruption and activate or deactivate raft-associated proteins, such as death receptor proteins, protein kinases, and calcium channels. Several anti-cancer drugs are able to suppress growth and induce apoptosis of tumor cells through alteration of lipid raft contents via disrupting lipid raft integrity. PMID:22289360

  9. Lipid raft: A floating island of death or survival.

    PubMed

    George, Kimberly S; Wu, Shiyong

    2012-03-15

    Lipid rafts are microdomains of the plasma membrane enriched in cholesterol and sphingolipids, and play an important role in the initiation of many pharmacological agent-induced signaling pathways and toxicological effects. The structure of lipid rafts is dynamic, resulting in an ever-changing content of both lipids and proteins. Cholesterol, as a major component of lipid rafts, is critical for the formation and configuration of lipid raft microdomains, which provide signaling platforms capable of activating both pro-apoptotic and anti-apoptotic signaling pathways. A change of cholesterol level can result in lipid raft disruption and activate or deactivate raft-associated proteins, such as death receptor proteins, protein kinases, and calcium channels. Several anti-cancer drugs are able to suppress growth and induce apoptosis of tumor cells through alteration of lipid raft contents via disrupting lipid raft integrity. PMID:22289360

  10. Lipid raft: A floating island of death or survival

    SciTech Connect

    George, Kimberly S.; Wu, Shiyong

    2012-03-15

    Lipid rafts are microdomains of the plasma membrane enriched in cholesterol and sphingolipids, and play an important role in the initiation of many pharmacological agent-induced signaling pathways and toxicological effects. The structure of lipid rafts is dynamic, resulting in an ever-changing content of both lipids and proteins. Cholesterol, as a major component of lipid rafts, is critical for the formation and configuration of lipid raft microdomains, which provide signaling platforms capable of activating both pro-apoptotic and anti-apoptotic signaling pathways. A change of cholesterol level can result in lipid raft disruption and activate or deactivate raft-associated proteins, such as death receptor proteins, protein kinases, and calcium channels. Several anti-cancer drugs are able to suppress growth and induce apoptosis of tumor cells through alteration of lipid raft contents via disrupting lipid raft integrity. -- Highlights: ► The role of lipid rafts in apoptosis ► The pro- and anti-apoptotic effects of lipid raft disruption ► Cancer treatments targeting lipid rafts.

  11. 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. PMID:27061495

  12. 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

  13. Cholesterol lipids and cholesterol-containing lipid rafts in bacteria.

    PubMed

    Huang, Zhen; London, Erwin

    2016-09-01

    Sterols are important components of eukaryotic membranes, but rare in bacteria. Some bacteria obtain sterols from their host or environment. In some cases, these sterols form membrane domains analogous the lipid rafts proposed to exist in eukaryotic membranes. This review describes the properties and roles of sterols in Borrelia and Helicobacter. PMID:26964703

  14. Lipid Rafts Establish Calcium Waves in Hepatocytes

    PubMed Central

    NAGATA, JUN; GUERRA, MATEUS T.; SHUGRUE, CHRISTINE A.; GOMES, DAWIDSON A.; NAGATA, NAOKI; NATHANSON, MICHAEL H.

    2010-01-01

    Background & Aims Polarity is critical for hepatocyte function. Ca2+ waves are polarized in hepatocytes because the inositol 1,4,5-trisphosphate receptor (InsP3R) is concentrated in the pericanalicular region, but the basis for this localization is unknown. We examined whether pericanalicular localization of the InsP3R and its action to trigger Ca2+ waves depends on lipid rafts. Methods Experiments were performed using isolated rat hepatocyte couplets and pancreatic acini, plus SkHep1 cells as nonpolarized controls. The cholesterol depleting agent methyl-beta-cyclodextrin (mβCD) was used to disrupt lipid rafts. InsP3R isoforms were examined by immunoblot and immunofluorescence. Ca2+ waves were examined by confocal microscopy. Results Type II InsP3Rs initially were localized to only some endoplasmic reticulum fractions in hepatocytes, but redistributed into all fractions in mβCD-treated cells. This InsP3R isoform was concentrated in the pericanalicular region, but redistributed throughout the cell after mβCD treatment. Vasopressin-induced Ca2+ signals began as apical-to-basal Ca2+ waves, and mβCD slowed the wave speed and prolonged the rise time. MβCD had a similar effect on Ca2+ waves in acinar cells but did not affect Ca2+ signals in SkHep1 cells, suggesting that cholesterol depletion has similar effects among polarized epithelia, but this is not a nonspecific effect of mβCD. Conclusions Lipid rafts are responsible for the pericanalicular accumulation of InsP3R in hepatocytes, and for the polarized Ca2+ waves that result. Signaling microdomains exist not only in the plasma membrane, but also in the nearby endoplasmic reticulum, which in turn, helps establish and maintain structural and functional polarity. PMID:17631147

  15. Lipid Rafts Disruption Increases Ochratoxin A Cytotoxicity to Hepatocytes.

    PubMed

    Zhang, Yu; Qi, Xiaozhe; Zheng, Juanjuan; Luo, Yunbo; Zhao, Changhui; Hao, Junran; Li, Xiaohong; Huang, Kunlun; Xu, Wentao

    2016-02-01

    Lipid rafts are microdomains in plasma membrane and can mediate cytotoxicity. In this study, the role of lipid rafts in ochratoxin A-induced toxicity was investigated using Hepatoblastoma Cell Line HepG-2 cells. Disruption of cholesterol-containing lipid rafts enhanced Ochratoxin A (OTA) toxicity, as shown by increased lactate dehydrogenase leakage, increased reactive oxygen species level and reduction of superoxide dismutase activity in a time-dependent manner. Isobaric tags for relative and absolute quantitation-based proteomics of the cell membranes showed that nearly 85.5% proteins were downregulated by OTA, indicating that OTA inhibited the membrane protein synthesis. Most of altered proteins were involved in Gene Ontology "transport", "cell adhesion" and "vesicle-mediated transport". In conclusion, lipid rafts play a key role in OTA-induced cytotoxicity. This study provides insight into how OTA toxicity is regulated by the plasma membrane, especially the lipid rafts. PMID:26861962

  16. Impact of Lipid Raft Integrity on 5-HT3 Receptor Function and its Modulation by Antidepressants

    PubMed Central

    Nothdurfter, Caroline; Tanasic, Sascha; Di Benedetto, Barbara; Rammes, Gerhard; Wagner, Eva-Maria; Kirmeier, Thomas; Ganal, Vanessa; Kessler, Julia S; Rein, Theo; Holsboer, Florian; Rupprecht, Rainer

    2010-01-01

    Because of the biochemical colocalization of the 5-HT3 receptor and antidepressants within raft-like domains and their antagonistic effects at this ligand-gated ion channel, we investigated the impact of lipid raft integrity for 5-HT3 receptor function and its modulation by antidepressants. Treatment with methyl-β-cyclodextrine (MβCD) markedly reduced membrane cholesterol levels and caused a more diffuse membrane distribution of the lipid raft marker protein flotillin-1 indicating lipid raft impairment. Both amplitude and charge of serotonin evoked cation currents were diminished following cholesterol depletion by either MβCD or simvastatin (Sim), whereas the functional antagonistic properties of the antidepressants desipramine (DMI) and fluoxetine (Fluox) at the 5-HT3 receptor were retained. Although both the 5-HT3 receptor and flotillin-1 were predominantly found in raft-like domains in western blots following sucrose density gradient centrifugation, immunocytochemistry revealed only a coincidental degree of colocalization of these two proteins. These findings and the persistence of the antagonistic effects of DMI and Fluox against 5-HT3 receptors after lipid raft impairment indicate that their modulatory effects are likely mediated through non-raft 5-HT3 receptors, which are not sufficiently detected by means of sucrose density gradient centrifugation. In conclusion, lipid raft integrity appears to be important for 5-HT3 receptor function in general, whereas it is not a prerequisite for the antagonistic properties of antidepressants such as DMI and Fluox at this ligand-gated ion channel. PMID:20200506

  17. Lipid rafts in immune signalling: current progress and future perspective.

    PubMed

    Varshney, Pallavi; Yadav, Vikas; Saini, Neeru

    2016-09-01

    Lipid rafts are dynamic assemblies of proteins and lipids that harbour many receptors and regulatory molecules and so act as a platform for signal transduction. They float freely within the liquid-disordered bilayer of cellular membranes and can cluster to form larger ordered domains. Alterations in lipid rafts are commonly found to be associated with the pathogenesis of several human diseases and recent reports have shown that the raft domains can also be perturbed by targeting raft proteins through microRNAs. Over the last few years, the importance of lipid rafts in modulating both innate and acquired immune responses has been elucidated. Various receptors present on immune cells like B cells, T cells, basophils and mast cells associate with lipid rafts on ligand binding and initiate signalling cascades leading to inflammation. Furthermore, disrupting lipid raft integrity alters lipopolysaccharide-induced cytokine secretion, IgE signalling, and B-cell and T-cell activation. The objective of this review is to summarize the recent progress in understanding the role of lipid rafts in the modulation of immune signalling and its related therapeutic potential for autoimmune diseases and inflammatory disorders. PMID:27153983

  18. Modifying lipid rafts promotes regeneration and functional recovery.

    PubMed

    Tassew, Nardos G; Mothe, Andrea J; Shabanzadeh, Alireza P; Banerjee, Paromita; Koeberle, Paulo D; Bremner, Rod; Tator, Charles H; Monnier, Philippe P

    2014-08-21

    Ideal strategies to ameliorate CNS damage should promote both neuronal survival and axon regeneration. The receptor Neogenin promotes neuronal apoptosis. Its ligand prevents death, but the resulting repulsive guidance molecule a (RGMa)-Neogenin interaction also inhibits axonal growth, countering any prosurvival benefits. Here, we explore strategies to inhibit Neogenin, thus simultaneously enhancing survival and regeneration. We show that bone morphogenetic protein (BMP) and RGMa-dependent recruitment of Neogenin into lipid rafts requires an interaction between RGMa and Neogenin subdomains. RGMa or Neogenin peptides that prevent this interaction, BMP inhibition by Noggin, or reduction of membrane cholesterol all block Neogenin raft localization, promote axon outgrowth, and prevent neuronal apoptosis. Blocking Neogenin raft association influences axonal pathfinding, enhances survival in the developing CNS, and promotes survival and regeneration in the injured adult optic nerve and spinal cord. Moreover, lowering cholesterol disrupts rafts and restores locomotor function after spinal cord injury. These data reveal a unified strategy to promote both survival and regeneration in the CNS. PMID:25127134

  19. Localization and signaling of GPCRs in lipid rafts.

    PubMed

    Villar, Van Anthony M; Cuevas, Santiago; Zheng, Xiaoxu; Jose, Pedro A

    2016-01-01

    The understanding of how biological membranes are organized and how they function has evolved. Instead of just serving as a medium in which certain proteins are found, portions of the lipid bilayer have been demonstrated to form specialized platforms that foster the assembly of signaling complexes by providing a microenvironment that is conducive for effective protein-protein interactions. G protein-coupled receptors (GPCRs) and relevant signaling molecules, including the heterotrimeric G proteins, key enzymes such as kinases and phosphatases, trafficking proteins, and secondary messengers, preferentially partition to these highly organized cell membrane microdomains, called lipid rafts. As such, lipid rafts are crucial for the trafficking and signaling of GPCRs. The study of GPCR biology in the context of lipid rafts involves the localization of the GPCR of interest in lipid rafts, at the basal state and upon receptor agonism, and the evaluation of the biological functions of the GPCR in appropriate cell lines. The lack of standardized methodology to study lipid rafts, in general, and of the workings of GPCRs in lipid rafts, in particular, and the inherent drawbacks of current methods have hampered the complete understanding of the underlying molecular mechanisms. Newer methodologies that allow the study of GPCRs in their native form are needed. The use of complementary approaches that produce mutually supportive results appear to be the best way for drawing conclusions with regards to the distribution and activity of GPCRs in lipid rafts. PMID:26928536

  20. Lipid raft involvement in yeast cell growth and death

    PubMed Central

    Mollinedo, Faustino

    2012-01-01

    The notion that cellular membranes contain distinct microdomains, acting as scaffolds for signal transduction processes, has gained considerable momentum. In particular, a class of such domains that is rich in sphingolipids and cholesterol, termed as lipid rafts, is thought to compartmentalize the plasma membrane, and to have important roles in survival and cell death signaling in mammalian cells. Likewise, yeast lipid rafts are membrane domains enriched in sphingolipids and ergosterol, the yeast counterpart of mammalian cholesterol. Sterol-rich membrane domains have been identified in several fungal species, including the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe as well as the pathogens Candida albicans and Cryptococcus neoformans. Yeast rafts have been mainly involved in membrane trafficking, but increasing evidence implicates rafts in a wide range of additional cellular processes. Yeast lipid rafts house biologically important proteins involved in the proper function of yeast, such as proteins that control Na+, K+, and pH homeostasis, which influence many cellular processes, including cell growth and death. Membrane raft constituents affect drug susceptibility, and drugs interacting with sterols alter raft composition and membrane integrity, leading to yeast cell death. Because of the genetic tractability of yeast, analysis of yeast rafts could be an excellent model to approach unanswered questions of mammalian raft biology, and to understand the role of lipid rafts in the regulation of cell death and survival in human cells. A better insight in raft biology might lead to envisage new raft-mediated approaches to the treatment of human diseases where regulation of cell death and survival is critical, such as cancer and neurodegenerative diseases. PMID:23087902

  1. Lipid rafts as major platforms for signaling regulation in cancer.

    PubMed

    Mollinedo, Faustino; Gajate, Consuelo

    2015-01-01

    Cell signaling does not apparently occur randomly over the cell surface, but it seems to be integrated very often into cholesterol-rich membrane domains, termed lipid rafts. Membrane lipid rafts are highly ordered membrane domains that are enriched in cholesterol, sphingolipids and gangliosides, and behave as major modulators of membrane geometry, lateral movement of molecules, traffic and signal transduction. Because the lipid and protein composition of membrane rafts differs from that of the surrounding membrane, they provide an additional level of compartmentalization, serving as sorting platforms and hubs for signal transduction proteins. A wide number of signal transduction processes related to cell adhesion, migration, as well as to cell survival and proliferation, which play major roles in cancer development and progression, are dependent on lipid rafts. Despite lipid rafts harbor mainly critical survival signaling pathways, including insulin-like growth factor I (IGF-I)/phosphatidylinositol 3-kinase (PI3K)/Akt signaling, recent evidence suggests that these membrane domains can also house death receptor-mediated apoptotic signaling. Recruitment of this death receptor signaling pathway in membrane rafts can be pharmacologically modulated, thus opening up the possibility to regulate cell demise with a therapeutic use. The synthetic ether phospholipid edelfosine shows a high affinity for cholesterol and accumulates in lipid rafts in a number of malignant hematological cells, leading to an efficient in vitro and in vivo antitumor activity by inducing translocation of death receptors and downstream signaling molecules to these membrane domains. Additional antitumor drugs have also been shown to act, at least in part, by recruiting death receptors in lipid rafts. The partition of death receptors together with downstream apoptotic signaling molecules in membrane rafts has led us to postulate the concept of a special liquid-ordered membrane platform coined as

  2. Dynamic Reorganization and Correlation among Lipid Raft Components.

    PubMed

    Lozano, Mónica M; Hovis, Jennifer S; Moss, Frank R; Boxer, Steven G

    2016-08-10

    Lipid rafts are widely believed to be an essential organizational motif in cell membranes. However, direct evidence for interactions among lipid and/or protein components believed to be associated with rafts is quite limited owing, in part, to the small size and intrinsically dynamic interactions that lead to raft formation. Here, we exploit the single negative charge on the monosialoganglioside GM1, commonly associated with rafts, to create a gradient of GM1 in response to an electric field applied parallel to a patterned supported lipid bilayer. The composition of this gradient is visualized by imaging mass spectrometry using a NanoSIMS. Using this analytical method, added cholesterol and sphingomyelin, both neutral and not themselves displaced by the electric field, are observed to reorganize with GM1. This dynamic reorganization provides direct evidence for an attractive interaction among these raft components into some sort of cluster. At steady state we obtain an estimate for the composition of this cluster. PMID:27447959

  3. Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes.

    PubMed

    Sezgin, Erdinc; Levental, Ilya; Grzybek, Michal; Schwarzmann, Günter; Mueller, Veronika; Honigmann, Alf; Belov, Vladimir N; Eggeling, Christian; Coskun, Unal; Simons, Kai; Schwille, Petra

    2012-07-01

    cellular plasma membranes consistently reveals a constant level of confined diffusion for raft lipid analogs that vary greatly in their partitioning behavior, suggesting different physicochemical bases for these phenomena. PMID:22450237

  4. Structure of Cholesterol in Lipid Rafts

    NASA Astrophysics Data System (ADS)

    Toppozini, Laura; Meinhardt, Sebastian; Armstrong, Clare L.; Yamani, Zahra; Kučerka, Norbert; Schmid, Friederike; Rheinstädter, Maikel C.

    2014-11-01

    Rafts, or functional domains, are transient nano-or mesoscopic structures in the plasma membrane and are thought to be essential for many cellular processes such as signal transduction, adhesion, trafficking, and lipid or protein sorting. Observations of these membrane heterogeneities have proven challenging, as they are thought to be both small and short lived. With a combination of coarse-grained molecular dynamics simulations and neutron diffraction using deuterium labeled cholesterol molecules, we observe raftlike structures and determine the ordering of the cholesterol molecules in binary cholesterol-containing lipid membranes. From coarse-grained computer simulations, heterogenous membranes structures were observed and characterized as small, ordered domains. Neutron diffraction was used to study the lateral structure of the cholesterol molecules. We find pairs of strongly bound cholesterol molecules in the liquid-disordered phase, in accordance with the umbrella model. Bragg peaks corresponding to ordering of the cholesterol molecules in the raftlike structures were observed and indexed by two different structures: a monoclinic structure of ordered cholesterol pairs of alternating direction in equilibrium with cholesterol plaques, i.e., triclinic cholesterol bilayers.

  5. The molecular face of lipid rafts in model membranes

    PubMed Central

    Risselada, H. Jelger; Marrink, Siewert J.

    2008-01-01

    Cell membranes contain a large number of different lipid species. Such a multicomponent mixture exhibits a complex phase behavior with regions of structural and compositional heterogeneity. Especially domains formed in ternary mixtures, composed of saturated and unsaturated lipids together with cholesterol, have received a lot of attention as they may resemble raft formation in real cells. Here we apply a simulation model to assess the molecular nature of these domains at the nanoscale, information that has thus far eluded experimental determination. We are able to show the spontaneous separation of a saturated phosphatidylcholine (PC)/unsaturated PC/cholesterol mixture into a liquid-ordered and a liquid-disordered phase with structural and dynamic properties closely matching experimental data. The near-atomic resolution of the simulations reveals remarkable features of both domains and the boundary domain interface. Furthermore, we predict the existence of a small surface tension between the monolayer leaflets that drives registration of the domains. At the level of molecular detail, raft-like lipid mixtures show a surprising face with possible implications for many cell membrane processes. PMID:18987307

  6. Identification of nucleolin as a lipid-raft-dependent β1-integrin-interacting protein in A375 cell migration.

    PubMed

    Bi, Jiajia; Wang, Ruifei; Zhang, Yue; Han, Xiaoqing; Ampah, Khamal Kwesi; Liu, Wenguang; Zeng, Xianlu

    2013-12-01

    Lipid rafts are related to cell surface receptor function. Integrin is a major surface receptor protein in cell adhesion and migration on the extracellular matrix (ECM). Here, we showed that lipid rafts played a critical role in human melanoma A375 cell spreading and migration on fibronectin; an important component of the ECM that interacts with β1 integrin. We found that the disruption of lipid rafts did not markedly inhibit the expression and activation of β1 integrin. By coimmunoprecipitation and mass spectrometry, we investigated the influence of lipid rafts on the β1 integrin complex and identified nucleolin as a potential lipid-raft-dependent β1-integrin-interacting protein. Upon confirmation of the interaction between β1 integrin and nucleolin, further studies revealed that nucleolin colocalized with β1 integrin in lipid rafts and raft disruption interrupted their association. In addition, knockdown of nucleolin markedly attenuated A375 cell spreading and migration on fibronectin. Taken together, we demonstrated that nucleolin is a critical lipid-raft-dependent β1-integrin-interacting protein in A375 cell spreading and migration on fibronectin. PMID:24292944

  7. Concerted diffusion of lipids in raft-like membranes.

    PubMed

    Apajalahti, Touko; Niemelä, Perttu; Govindan, Praveen Nedumpully; Miettinen, Markus S; Salonen, Emppu; Marrink, Siewert-Jan; Vattulainen, Ilpo

    2010-01-01

    Currently, there is no comprehensive model for the dynamics of cellular membranes. The understanding of even the basic dynamic processes, such as lateral diffusion of lipids, is still quite limited. Recent studies of one-component membrane systems have shown that instead of single-particle motions, the lateral diffusion is driven by a more complex, concerted mechanism for lipid diffusion (E. Falck et al., J. Am. Chem. Soc., 2008, 130, 44-45), where a lipid and its neighbors move in unison in terms of loosely defined clusters. In this work, we extend the previous study by considering the concerted lipid diffusion phenomena in many-component raft-like membranes. This nature of diffusion phenomena emerge in all the cases we have considered, including both atom-scale simulations of lateral diffusion within rafts and coarse-grained MARTINI simulations of diffusion in membranes characterized by coexistence of raft and non-raft domains. The data allows us to identify characteristic time scales for the concerted lipid motions, which turn out to range from hundreds of nanoseconds to several microseconds. Further, we characterize typical length scales associated with the correlated lipid diffusion patterns and find them to be about 10 nm, or even larger if weak correlations are taken into account. Finally, the concerted nature of lipid motions is also found in dissipative particle dynamics simulations of lipid membranes, clarifying the role of hydrodynamics (local momentum conservation) in membrane diffusion phenomena. PMID:20158041

  8. 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. PMID:25553454

  9. Monolayer curvature stabilizes nanoscale raft domains in mixed lipid bilayers

    PubMed Central

    Meinhardt, Sebastian; Vink, Richard L. C.; Schmid, Friederike

    2013-01-01

    According to the lipid raft hypothesis, biological lipid membranes are laterally heterogeneous and filled with nanoscale ordered “raft” domains, which are believed to play an important role for the organization of proteins in membranes. However, the mechanisms stabilizing such small rafts are not clear, and even their existence is sometimes questioned. Here, we report the observation of raft-like structures in a coarse-grained molecular model for multicomponent lipid bilayers. On small scales, our membranes demix into a liquid ordered (lo) phase and a liquid disordered (ld) phase. On large scales, phase separation is suppressed and gives way to a microemulsion-type state that contains nanometer-sized lo domains in an ld environment. Furthermore, we introduce a mechanism that generates rafts of finite size by a coupling between monolayer curvature and local composition. We show that mismatch between the spontaneous curvatures of monolayers in the lo and ld phases induces elastic interactions, which reduce the line tension between the lo and ld phases and can stabilize raft domains with a characteristic size of the order of a few nanometers. Our findings suggest that rafts in multicomponent bilayers might be closely related to the modulated ripple phase in one-component bilayers. PMID:23487780

  10. 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

  11. Thermally induced changes in lipid composition of raft and non-raft regions of hepatocyte plasma membranes of rainbow trout.

    PubMed

    Zehmer, John K; Hazel, Jeffrey R

    2005-11-01

    In poikilotherms, increases in plasma membrane (PM) cholesterol and an increase in the degree of lipid acyl chain saturation commonly accompany an increase in growth temperature. This has typically been interpreted in terms of membrane fluidity/order homeostasis, but these changes would also be expected to stabilize the structure of PM rafts against thermal perturbation. Rafts are microdomains that organize the molecules of many signaling cascades and are formed as a result of interactions between lipids with saturated acyl chains and cholesterol. No study to date has examined the thermally induced compositional changes of raft and non-raft regions of the PM separately. In this study we have measured the phospholipid class composition and fatty acid composition of raft-enriched (raft) and raft-depleted PM (RDPM) of hepatocytes from trout Oncorhynchus mykiss acclimated to 5 degrees C and 20 degrees C. In the raft, warm acclimation was associated with a reduction in the proportion of phosphatidylcholine from 56% to 30% while phosphatidylserine and phosphatidylinositol each increased from 8% to approximately 20% of the total phospholipid. Additionally, there were significantly fewer unsaturated fatty acids in the raft lipids from warm-acclimated (61%) than from the cold-acclimated trout (68%). In contrast, there were no significant changes in phospholipid class or acyl chain unsaturation in the RDPM. These data suggest that changes in raft lipid composition, rather than the PM as a whole, are particularly important during thermal acclimation. PMID:16272251

  12. Raft lipids as common components of human extracellular amyloid fibrils

    PubMed Central

    Gellermann, Gerald P.; Appel, Thomas R.; Tannert, Astrid; Radestock, Anja; Hortschansky, Peter; Schroeckh, Volker; Leisner, Christian; Lütkepohl, Tim; Shtrasburg, Shmuel; Röcken, Christoph; Pras, Mordechai; Linke, Reinhold P.; Diekmann, Stephan; Fändrich, Marcus

    2005-01-01

    Amyloid fibrils are fibrillar polypeptide aggregates from several degenerative human conditions, including Alzheimer's and Creutzfeldt-Jakob diseases. Analysis of amyloid fibrils derived from various human diseases (AA, ATTR, Aβ2M, ALλ, and ALκ amyloidosis) shows that these are associated with a common lipid component that has a conserved chemical composition and that is specifically rich in cholesterol and sphingolipids, the major components of cellular lipid rafts. This pattern is not notably affected by the purification procedure, and no tight lipid interactions can be detected when preformed fibrils are mixed with lipids. By contrast, the early and prefibrillar aggregates formed in an AA amyloid-producing cell system interact with the raft marker ganglioside-1, and amyloid formation is impaired by addition of cholesterol-reducing agents. These data suggest the existence of common cellular mechanisms in the generation of different types of clinical amyloid deposits. PMID:15851687

  13. 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

  14. Caveolin interaction governs Kv1.3 lipid raft targeting

    PubMed Central

    Pérez-Verdaguer, Mireia; Capera, Jesusa; Martínez-Mármol, Ramón; Camps, Marta; Comes, Núria; Tamkun, Michael M.; Felipe, Antonio

    2016-01-01

    The spatial localization of ion channels at the cell surface is crucial for their functional role. Many channels localize in lipid raft microdomains, which are enriched in cholesterol and sphingolipids. Caveolae, specific lipid rafts which concentrate caveolins, harbor signaling molecules and their targets becoming signaling platforms crucial in cell physiology. However, the molecular mechanisms involved in such spatial localization are under debate. Kv1.3 localizes in lipid rafts and participates in the immunological response. We sought to elucidate the mechanisms of Kv1.3 surface targeting, which govern leukocyte physiology. Kv1 channels share a putative caveolin-binding domain located at the intracellular N-terminal of the channel. This motif, lying close to the S1 transmembrane segment, is situated near the T1 tetramerization domain and the determinants involved in the Kvβ subunit association. The highly hydrophobic domain (FQRQVWLLF) interacts with caveolin 1 targeting Kv1.3 to caveolar rafts. However, subtle variations of this cluster, putative ancillary associations and different structural conformations can impair the caveolin recognition, thereby altering channel’s spatial localization. Our results identify a caveolin-binding domain in Kv1 channels and highlight the mechanisms that govern the regulation of channel surface localization during cellular processes. PMID:26931497

  15. Caveolin interaction governs Kv1.3 lipid raft targeting.

    PubMed

    Pérez-Verdaguer, Mireia; Capera, Jesusa; Martínez-Mármol, Ramón; Camps, Marta; Comes, Núria; Tamkun, Michael M; Felipe, Antonio

    2016-01-01

    The spatial localization of ion channels at the cell surface is crucial for their functional role. Many channels localize in lipid raft microdomains, which are enriched in cholesterol and sphingolipids. Caveolae, specific lipid rafts which concentrate caveolins, harbor signaling molecules and their targets becoming signaling platforms crucial in cell physiology. However, the molecular mechanisms involved in such spatial localization are under debate. Kv1.3 localizes in lipid rafts and participates in the immunological response. We sought to elucidate the mechanisms of Kv1.3 surface targeting, which govern leukocyte physiology. Kv1 channels share a putative caveolin-binding domain located at the intracellular N-terminal of the channel. This motif, lying close to the S1 transmembrane segment, is situated near the T1 tetramerization domain and the determinants involved in the Kvβ subunit association. The highly hydrophobic domain (FQRQVWLLF) interacts with caveolin 1 targeting Kv1.3 to caveolar rafts. However, subtle variations of this cluster, putative ancillary associations and different structural conformations can impair the caveolin recognition, thereby altering channel's spatial localization. Our results identify a caveolin-binding domain in Kv1 channels and highlight the mechanisms that govern the regulation of channel surface localization during cellular processes. PMID:26931497

  16. Lipid rafts are required for Kit survival and proliferation signals

    PubMed Central

    Leifheit, Erica; Gooch, Stacie; Sindhu, Simran; Weinberg, Kenneth

    2007-01-01

    In addition to its physiologic role as central regulator of the hematopoietic and reproductive systems, the Kit receptor tyrosine kinase (RTK) is pathologically overexpressed in some forms of leukemia and constitutively activated by oncogenic mutations in mast-cell proliferations and gastrointestinal stromal tumors. To gain insight into the general activation and signaling mechanisms of RTKs, we investigated the activation-dependent dynamic membrane distributions of wild-type and oncogenic forms of Kit in hematopoietic cells. Ligand-induced recruitment of wild-type Kit to lipid rafts after stimulation by Kit ligand (KL) and the constitutive localization of oncogenic Kit in lipid rafts are necessary for Kit-mediated proliferation and survival signals. KL-dependent and oncogenic Kit kinase activity resulted in recruitment of the regulatory phosphatidylinositol 3-kinase (PI3-K) subunit p85 to rafts where the catalytical PI3-K subunit p110 constitutively resides. Cholesterol depletion by methyl-β-cyclodextrin prevented Kit-mediated activation of the PI3-K downstream target Akt and inhibited cellular proliferation by KL-activated or oncogenic Kit, including mutants resistant to the Kit inhibitor imatinib-mesylate. Our data are consistent with the notion that Kit recruitment to lipid rafts is required for efficient activation of the PI3-K/Akt pathway and Kit-mediated proliferation. PMID:17554062

  17. Lipid rafts direct macrophage motility in the tissue microenvironment.

    PubMed

    Previtera, Michelle L; Peterman, Kimberly; Shah, Smit; Luzuriaga, Juan

    2015-04-01

    Infiltrating leukocytes are exposed to a wide range of tissue elasticities. While we know the effects of substrate elasticity on acute inflammation via the study of neutrophil migration, we do not know its effects on leukocytes that direct chronic inflammatory events. Here, we studied morphology and motility of macrophages, the innate immune cells that orchestrate acute and chronic inflammation, on polyacrylamide hydrogels that mimicked a wide range of tissue elasticities. As expected, we found that macrophage spreading area increased as substrate elasticity increased. Unexpectedly, we found that morphology did not inversely correlate with motility. In fact, velocity of steady-state macrophages remained unaffected by substrate elasticity, while velocity of biologically stimulated macrophages was limited on stiff substrates. We also found that the lack of motility on stiff substrates was due to a lack of lipid rafts on the leading edge of the macrophages. This study implicates lipid rafts in the mechanosensory mechanism of innate immune cell infiltration. PMID:25269613

  18. Functional role of lipid rafts in CD20 activity?

    PubMed

    Janas, Eva; Priest, Richard; Malhotra, Rajneesh

    2005-01-01

    CD20 is a B-lymphocyte-specific integral membrane protein, implicated in the regulation of transmembrane calcium conductance, cell-cycle progression and B-lymphocyte proliferation. CD20 is proposed to function as a SOCC (store-operated calcium channel). SOCCs are activated by receptor-stimulated calcium depletion of intracellular stores. Sustained calcium conductivity across the plasma membrane mediated by SOCC activity is required for long-term calcium-dependent processes, such as transcriptional control and gene expression. Cross-linking of CD20 by antibodies (e.g. Rituxan) has been reported to induce a rapid redistribution of CD20 into specialized microdomains at the plasma membrane, known as lipid rafts. Recruitment of CD20 into lipid rafts and its homo-oligomerization are suggested to be crucial for CD20 activity and regulation. This review outlines recent biochemical studies characterizing the role of CD20 in calcium signalling in B-lymphocytes and evaluates an engagement of lipid rafts in the regulation of CD20-mediated calcium conductivity. PMID:15649140

  19. Ethanol Enhances TGF-β Activity by Recruiting TGF-β Receptors From Intracellular Vesicles/Lipid Rafts/Caveolae to Non-Lipid Raft Microdomains.

    PubMed

    Huang, Shuan Shian; Chen, Chun-Lin; Huang, Franklin W; Johnson, Frank E; Huang, Jung San

    2016-04-01

    Regular consumption of moderate amounts of ethanol has important health benefits on atherosclerotic cardiovascular disease (ASCVD). Overindulgence can cause many diseases, particularly alcoholic liver disease (ALD). The mechanisms by which ethanol causes both beneficial and harmful effects on human health are poorly understood. Here we demonstrate that ethanol enhances TGF-β-stimulated luciferase activity with a maximum of 0.5-1% (v/v) in Mv1Lu cells stably expressing a luciferase reporter gene containing Smad2-dependent elements. In Mv1Lu cells, 0.5% ethanol increases the level of P-Smad2, a canonical TGF-β signaling sensor, by ∼2-3-fold. Ethanol (0.5%) increases cell-surface expression of the type II TGF-β receptor (TβR-II) by ∼2-3-fold from its intracellular pool, as determined by I(125) -TGF-β-cross-linking/Western blot analysis. Sucrose density gradient ultracentrifugation and indirect immunofluorescence staining analyses reveal that ethanol (0.5% and 1%) also displaces cell-surface TβR-I and TβR-II from lipid rafts/caveolae and facilitates translocation of these receptors to non-lipid raft microdomains where canonical signaling occurs. These results suggest that ethanol enhances canonical TGF-β signaling by increasing non-lipid raft microdomain localization of the TGF-β receptors. Since TGF-β plays a protective role in ASCVD but can also cause ALD, the TGF-β enhancer activity of ethanol at low and high doses appears to be responsible for both beneficial and harmful effects. Ethanol also disrupts the location of lipid raft/caveolae of other membrane proteins (e.g., neurotransmitter, growth factor/cytokine, and G protein-coupled receptors) which utilize lipid rafts/caveolae as signaling platforms. Displacement of these membrane proteins induced by ethanol may result in a variety of pathologies in nerve, heart and other tissues. J. Cell. Biochem. 117: 860-871, 2016. © 2015 Wiley Periodicals, Inc. PMID:26419316

  20. 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

    2016-07-01

    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

  1. Proteomic Characterization of Lipid Raft Proteins in ALS Mouse Spinal Cord

    PubMed Central

    Zhai, Jianjun; Ström, Anna-Lena; Kilty, Renee; Venkatakrishnan, Priya; White, James; Everson, William V.; Smart, Eric J.; Zhu, Haining

    2009-01-01

    Summary Familial amyotrophic lateral sclerosis (ALS) has been linked to mutations in the copper/zinc superoxide dismutase (SOD1) gene. The mutant SOD1 protein exhibits a toxic gain-of-function that adversely affects the function of neurons. However, the mechanism of how mutant SOD1 initiates ALS is unclear. Lipid rafts are specialized microdomains of the plasma membrane that act as platforms for the organization and interaction of proteins involved in multiple functions including vesicular trafficking, neurotransmitter signaling and cytoskeletal rearrangements. In this study, we report a proteomic analysis using a widely used ALS mouse model to identify differences in spinal cord lipid raft proteomes between mice over-expressing wild-type (WT) and G93A mutant SOD1. A total of 413 and 421 proteins were identified in the lipid rafts isolated from WT and G93A mice, respectively. Further quantitative analysis revealed a consortium of proteins with altered levels between the WT and G93A samples. Functional classification of the 67 altered proteins revealed that the three most impacted subsets of proteins were involved in vesicular transport, neurotransmitter synthesis and release; cytoskeleton organization and linkage to plasma membrane; and metabolism. Other protein changes are correlated with alterations in microglia activation and inflammation; astrocyte and oligodendrocyte function; cell signaling; cellular stress response and apoptosis; and neuronal ion channels and neurotransmitter receptor functions. Changes of selected proteins were independently validated by immunoblotting and immunohistochemistry. The significance of the lipid raft protein changes in motor neuron function and degeneration in ALS is discussed, particularly those involved in vesicular trafficking and neurotransmitter signaling, and the dynamics and regulation of plasma membrane-anchored cytoskeleton. PMID:19438725

  2. Proteomic characterization of lipid raft proteins in amyotrophic lateral sclerosis mouse spinal cord.

    PubMed

    Zhai, Jianjun; Ström, Anna-Lena; Kilty, Renee; Venkatakrishnan, Priya; White, James; Everson, William V; Smart, Eric J; Zhu, Haining

    2009-06-01

    Familial amyotrophic lateral sclerosis (ALS) has been linked to mutations in the copper/zinc superoxide dismutase (SOD1) gene. The mutant SOD1 protein exhibits a toxic gain-of-function that adversely affects the function of neurons. However, the mechanism by which mutant SOD1 initiates ALS is unclear. Lipid rafts are specialized microdomains of the plasma membrane that act as platforms for the organization and interaction of proteins involved in multiple functions, including vesicular trafficking, neurotransmitter signaling, and cytoskeletal rearrangements. In this article, we report a proteomic analysis using a widely used ALS mouse model to identify differences in spinal cord lipid raft proteomes between mice overexpressing wild-type (WT) and G93A mutant SOD1. In total, 413 and 421 proteins were identified in the lipid rafts isolated from WT and G93A mice, respectively. Further quantitative analysis revealed a consortium of proteins with altered levels between the WT and G93A samples. Functional classification of the 67 altered proteins revealed that the three most affected subsets of proteins were involved in: vesicular transport, and neurotransmitter synthesis and release; cytoskeletal organization and linkage to the plasma membrane; and metabolism. Other protein changes were correlated with alterations in: microglia activation and inflammation; astrocyte and oligodendrocyte function; cell signaling; cellular stress response and apoptosis; and neuronal ion channels and neurotransmitter receptor functions. Changes of selected proteins were independently validated by immunoblotting and immunohistochemistry. The significance of the lipid raft protein changes in motor neuron function and degeneration in ALS is discussed, particularly for proteins involved in vesicular trafficking and neurotransmitter signaling, and the dynamics and regulation of the plasma membrane-anchored cytoskeleton. PMID:19438725

  3. Dynamic clustering and dispersion of lipid rafts contribute to fusion competence of myogenic cells

    SciTech Connect

    Mukai, Atsushi; Kurisaki, Tomohiro; Sato, Satoshi B.; Kobayashi, Toshihide; Kondoh, Gen; Hashimoto, Naohiro

    2009-10-15

    Recent research indicates that the leading edge of lamellipodia of myogenic cells (myoblasts and myotubes) contains presumptive fusion sites, yet the mechanisms that render the plasma membrane fusion-competent remain largely unknown. Here we show that dynamic clustering and dispersion of lipid rafts contribute to both cell adhesion and plasma membrane union during myogenic cell fusion. Adhesion-complex proteins including M-cadherin, {beta}-catenin, and p120-catenin accumulated at the leading edge of lamellipodia, which contains the presumptive fusion sites of the plasma membrane, in a lipid raft-dependent fashion prior to cell contact. In addition, disruption of lipid rafts by cholesterol depletion directly prevented the membrane union of myogenic cell fusion. Time-lapse recording showed that lipid rafts were laterally dispersed from the center of the lamellipodia prior to membrane fusion. Adhesion proteins that had accumulated at lipid rafts were also removed from the presumptive fusion sites when lipid rafts were laterally dispersed. The resultant lipid raft- and adhesion complex-free area at the leading edge fused with the opposing plasma membrane. These results demonstrate a key role for dynamic clustering/dispersion of lipid rafts in establishing fusion-competent sites of the myogenic cell membrane, providing a novel mechanistic insight into the regulation of myogenic cell fusion.

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

    SciTech Connect

    Thayanithy, Venugopal; Babatunde, Victor; Dickson, Elizabeth L.; Wong, Phillip; Oh, Sanghoon; Ke, Xu; Barlas, Afsar; Fujisawa, Sho; Romin, Yevgeniy; Moreira, André L.; Downey, Robert J.; Steer, Clifford J.; Subramanian, Subbaya; Manova-Todorova, Katia; Moore, Malcolm A.S.; Lou, Emil

    2014-04-15

    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 formation (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 and

  5. Lipid rafts-mediated endocytosis and physiology-based cell membrane traffic models of doxorubicin liposomes.

    PubMed

    Li, Yinghuan; Gao, Lei; Tan, Xi; Li, Feiyang; Zhao, Ming; Peng, Shiqi

    2016-08-01

    The clathrin-mediated endocytosis is likely a major mechanism of liposomes' internalization. A kinetic approach was used to assess the internalization mechanism of doxorubicin (Dox) loaded cationic liposomes and to establish physiology-based cell membrane traffic mathematic models. Lipid rafts-mediated endocytosis, including dynamin-dependent or -independent endocytosis of noncaveolar structure, was a dominant process. The mathematic models divided Dox loaded liposomes binding lipid rafts (B) into saturable binding (SB) and nonsaturable binding (NSB) followed by energy-driven endocytosis. The intracellular trafficking demonstrated early endosome-late endosome-lysosome or early/late endosome-cytoplasm-nucleus pathways. The three properties of liposome structures, i.e., cationic lipid, fusogenic lipid, and pegylation, were investigated to compare their contributions to cell membrane and intracellular traffic. The results revealed great contribution of cationic lipid DOTAP and fusogenic lipid DOPE to cell membrane binding and internalization. The valid Dox in the nuclei of HepG2 and A375 cells treated with cationic liposomes containing 40mol% of DOPE were 1.2-fold and 1.5-fold higher than that in the nuclei of HepG2 and A375 cells treated with liposomes containing 20mol% of DOPE, respectively, suggesting the dependence of cell type. This tendency was proportional to the increase of cell-associated total liposomal Dox. The mathematic models would be useful to predict intracellular trafficking of liposomal Dox. PMID:27117641

  6. Prion Protein Accumulation in Lipid Rafts of Mouse Aging Brain

    PubMed Central

    Agostini, Federica; Dotti, Carlos G.; Pérez-Cañamás, Azucena; Ledesma, Maria Dolores; Benetti, Federico; Legname, Giuseppe

    2013-01-01

    The cellular form of the prion protein (PrPC) is a normal constituent of neuronal cell membranes. The protein misfolding causes rare neurodegenerative disorders known as transmissible spongiform encephalopathies or prion diseases. These maladies can be sporadic, genetic or infectious. Sporadic prion diseases are the most common form mainly affecting aging people. In this work, we investigate the biochemical environment in which sporadic prion diseases may develop, focusing our attention on the cell membrane of neurons in the aging brain. It is well established that with aging the ratio between the most abundant lipid components of rafts undergoes a major change: while cholesterol decreases, sphingomyelin content rises. Our results indicate that the aging process modifies the compartmentalization of PrPC. In old mice, this change favors PrPC accumulation in detergent-resistant membranes, particularly in hippocampi. To confirm the relationship between lipid content changes and PrPC translocation into detergent-resistant membranes (DRMs), we looked at PrPC compartmentalization in hippocampi from acid sphingomyelinase (ASM) knockout (KO) mice and synaptosomes enriched in sphingomyelin. In the presence of high sphingomyelin content, we observed a significant increase of PrPC in DRMS. This process is not due to higher levels of total protein and it could, in turn, favor the onset of sporadic prion diseases during aging as it increases the PrP intermolecular contacts into lipid rafts. We observed that lowering sphingomyelin in scrapie-infected cells by using fumonisin B1 led to a 50% decrease in protease-resistant PrP formation. This may suggest an involvement of PrP lipid environment in prion formation and consequently it may play a role in the onset or development of sporadic forms of prion diseases. PMID:24040215

  7. 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

  8. Lipid rafts regulate cellular CD40 receptor localization in vascular endothelial cells

    SciTech Connect

    Xia Min; Wang Qing; Zhu Huilian; Ma Jing; Hou Mengjun; Tang Zhihong; Li Juanjuan; Ling Wenhua

    2007-09-28

    Cholesterol enriched lipid rafts are considered to function as platforms involved in the regulation of membrane receptor signaling complex through the clustering of signaling molecules. In this study, we tested whether these specialized membrane microdomains affect CD40 localization in vitro and in vivo. Here, we provide evidence that upon CD40 ligand stimulation, endogenous and exogenous CD40 receptor is rapidly mobilized into lipid rafts compared with unstimulated HAECs. Efficient binding between CD40L and CD40 receptor also increases amounts of CD40 protein levels in lipid rafts. Deficiency of intracellular conserved C terminus of the CD40 cytoplasmic tail impairs CD40 partitioning in raft. Raft disorganization after methyl-{beta}-cyclodextrin treatment diminishes CD40 localization into rafts. In vivo studies show that elevation of circulating cholesterol in high-cholesterol fed rabbits increases the cholesterol content and CD40 receptor localization in lipid rafts. These findings identify a physiological role for membrane lipid rafts as a critical regulator of CD40-mediated signal transduction and raise the possibility that certain pathologic conditions may be treated by altering CD40 signaling with drugs affecting its raft localization.

  9. 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

  10. Omega-3 fatty acids, lipid rafts, and T cell signaling.

    PubMed

    Hou, Tim Y; McMurray, David N; Chapkin, Robert S

    2016-08-15

    n-3 polyunsaturated fatty acids (PUFA) have been shown in many clinical studies to attenuate inflammatory responses. Although inflammatory responses are orchestrated by a wide spectrum of cells, CD4(+) T cells play an important role in the etiology of many chronic inflammatory diseases such as inflammatory bowel disease and obesity. In light of recent concerns over the safety profiles of non-steroidal anti-inflammatory drugs (NSAIDs), alternatives such as bioactive nutraceuticals are becoming more attractive. In order for these agents to be accepted into mainstream medicine, however, the mechanisms by which nutraceuticals such as n-3 PUFA exert their anti-inflammatory effects must be fully elucidated. Lipid rafts are nanoscale, dynamic domains in the plasma membrane that are formed through favorable lipid-lipid (cholesterol, sphingolipids, and saturated fatty acids) and lipid-protein (membrane-actin cytoskeleton) interactions. These domains optimize the clustering of signaling proteins at the membrane to facilitate efficient cell signaling which is required for CD4(+) T cell activation and differentiation. This review summarizes novel emerging data documenting the ability of n-3 PUFA to perturb membrane-cytoskeletal structure and function in CD4(+) T cells. An understanding of these underlying mechanisms will provide a rationale for the use of n-3 PUFA in the treatment of chronic inflammation. PMID:26001374

  11. Evidence for the involvement of lipid rafts localized at the ER-mitochondria associated membranes in autophagosome formation.

    PubMed

    Garofalo, Tina; Matarrese, Paola; Manganelli, Valeria; Marconi, Matteo; Tinari, Antonella; Gambardella, Lucrezia; Faggioni, Alberto; Misasi, Roberta; Sorice, Maurizio; Malorni, Walter

    2016-06-01

    Mitochondria-associated membranes (MAMs) are subdomains of the endoplasmic reticulum (ER) that interact with mitochondria. This membrane scrambling between ER and mitochondria appears to play a critical role in the earliest steps of autophagy. Recently, lipid microdomains, i.e. lipid rafts, have been identified as further actors of the autophagic process. In the present work, a series of biochemical and molecular analyses has been carried out in human fibroblasts with the specific aim of characterizing lipid rafts in MAMs and to decipher their possible implication in the autophagosome formation. In fact, the presence of lipid microdomains in MAMs has been detected and, in these structures, a molecular interaction of the ganglioside GD3, a paradigmatic "brick" of lipid rafts, with core-initiator proteins of autophagy, such as AMBRA1 and WIPI1, was revealed. This association seems thus to take place in the early phases of autophagic process in which MAMs have been hypothesized to play a key role. The functional activity of GD3 was suggested by the experiments carried out by knocking down ST8SIA1 gene expression, i.e., the synthase that leads to the ganglioside formation. This experimental condition results in fact in the impairment of the ER-mitochondria crosstalk and the subsequent hindering of autophagosome nucleation. We thus hypothesize that MAM raft-like microdomains could be pivotal in the initial organelle scrambling activity that finally leads to the formation of autophagosome. PMID:27123544

  12. Ginsenoside Rh2 induces ligand-independent Fas activation via lipid raft disruption

    SciTech Connect

    Yi, Jae-Sung; Choo, Hyo-Jung; Cho, Bong-Rae; Kim, Hwan-Myung; Kim, Yong-Nyun; Ham, Young-Mi; Ko, Young-Gyu

    2009-07-24

    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, 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.

  13. Selective Association of Outer Surface Lipoproteins with the Lipid Rafts of Borrelia burgdorferi

    PubMed Central

    Toledo, Alvaro; Crowley, Jameson T.; Coleman, James L.; LaRocca, Timothy J.; Chiantia, Salvatore; London, Erwin; Benach, Jorge L.

    2014-01-01

    ABSTRACT Borrelia burgdorferi contains unique cholesterol-glycolipid-rich lipid rafts that are associated with lipoproteins. These complexes suggest the existence of macromolecular structures that have not been reported for prokaryotes. Outer surface lipoproteins OspA, OspB, and OspC were studied for their participation in the formation of lipid rafts. Single-gene deletion mutants with deletions of ∆ospA, ∆ospB, and ∆ospC and a spontaneous gene mutant, strain B313, which does not express OspA and OspB, were used to establish their structural roles in the lipid rafts. All mutant strains used in this study produced detergent-resistant membranes, a common characteristic of lipid rafts, and had similar lipid and protein slot blot profiles. Lipoproteins OspA and OspB but not OspC were shown to be associated with lipid rafts by transmission electron microscopy. When the ability to form lipid rafts in live B. burgdorferi spirochetes was measured by fluorescence resonance energy transfer (FRET), strain B313 showed a statistically significant lower level of segregation into ordered and disordered membrane domains than did the wild-type and the other single-deletion mutants. The transformation of a B313 strain with a shuttle plasmid containing ospA restored the phenotype shared by the wild type and the single-deletion mutants, demonstrating that OspA and OspB have redundant functions. In contrast, a transformed B313 overexpressing OspC neither rescued the FRET nor colocalized with the lipid rafts. Because these lipoproteins are expressed at different stages of the life cycle of B. burgdorferi, their selective association is likely to have an important role in the structure of prokaryotic lipid rafts and in the organism’s adaptation to changing environments. PMID:24618252

  14. Herpes simplex virus protein UL11 but not UL51 is associated with lipid rafts.

    PubMed

    Koshizuka, Tetsuo; Kawaguchi, Yasushi; Nozawa, Naoki; Mori, Isamu; Nishiyama, Yukihiro

    2007-12-01

    The UL11 and UL51 gene products of herpes simplex virus (HSV) are membrane-associated tegument proteins that are incorporated into the HSV virion. UL11 and UL51 are conserved throughout the herpesvirus family. Both UL11 and UL51, either singly or in combination, are involved in virion envelopment and/or egress. Both proteins are fatty acylated: UL11 is both acylated by myristoic and palmitoic acids and UL51 is monoacylated by palmitoic acid. Using confocal microscopy and sucrose gradient fractionations in transfected or HSV-infected cells, we found that HSV-2 UL11 but not UL51 was associated with lipid rafts. The dual acylation of UL11 was necessary for lipid raft association, as mutations in the myristoylation or palmitoylation sites prevented lipid raft association. These differences in lipid raft association may contribute to the functional differences between UL11 and UL51. PMID:17694428

  15. Simvastatin Attenuates Astrogliosis after Traumatic Brain Injury through the Modulation of EGFR in Lipid Rafts

    PubMed Central

    Wu, Hongtao; Mahmood, Asim; Lu, Dunyue; Jiang, Hao; Xiong, Ye; Zhou, Dong; Chopp, Michael

    2010-01-01

    Objective Our previous studies demonstrated that simvastatin treatment promotes neuronal survival and reduces inflammatory cytokine release from astrocytes after traumatic brain injury (TBI) in rats. Since reactive astrocytes produce inflammation mediators, in the current study we investigated the effect of simvastatin on astrocyte activation after TBI and its underlying signaling mechanisms. Methods Saline or simvastatin (1 mg/kg) was orally administered to rats starting at Day 1 after TBI and then daily for 14 days. Rats were sacrificed at 1, 3, 7, 14 days after treatment. Brain sections and tissues were prepared for immunohistochemical staining and Western blot analysis, respectively. Cultured astrocytes were subjected to oxygen-glucose deprivation (OGD) and followed by immunocytochemical staining with GFAP/caveolin-1 and Western blot analysis. Lipid rafts were isolated from the cell lysate and Western blot was carried out to detect the changes in epidermal growth factor receptor (EGFR) expression and phosphorylation in the lipid rafts. Results Simvastatin significantly promoted neuronal survival after TBI and attenuated activation of astrocytes. Simvastatin modified the caveolin-1 expression in lipid rafts in astrocyte cell membrane, suppressed the phosphorylation of EGFR in lipid rafts of astrocytes after OGD, and inhibited the OGD-induced interleukin-1 (IL-1) production. Conclusions These data suggest that simvastatin reduces reactive astrogliosis and rescues neuronal cells after TBI. These beneficial effects of simvastatin may be mediated by inhibiting astrocyte activation after TBI through modifying the caveolin-1 expression in lipid rafts and the subsequent modulation of EGFR phosphorylation in lipid rafts. PMID:19895202

  16. The shedding activity of ADAM17 is sequestered in lipid rafts

    SciTech Connect

    Tellier, Edwige; Canault, Matthias; Rebsomen, Laure; Bonardo, Bernadette; Juhan-Vague, Irene; Nalbone, Gilles; Peiretti, Franck . E-mail: Franck.Peiretti@medecine.univ-mrs.fr

    2006-12-10

    The tumor necrosis factor-alpha (TNF) converting enzyme (ADAM17) is a metalloprotease-disintegrin responsible for the cleavage of several biologically active transmembrane proteins. However, the substrate specificity of ADAM17 and the regulation of its shedding activity are still poorly understood. Here, we report that during its transport through the Golgi apparatus, ADAM17 is included in cholesterol-rich membrane microdomains (lipid rafts) where its prodomain is cleaved by furin. Consequently, ADAM17 shedding activity is sequestered in lipid rafts, which is confirmed by the fact that metalloproteinase inhibition increases the proportion of ADAM17 substrates (TNF and its receptors TNFR1 and TNFR2) in lipid rafts. Membrane cholesterol depletion increases the ADAM17-dependent shedding of these substrates demonstrating the importance of lipid rafts in the control of this process. Furthermore, ADAM17 substrates are present in different proportions in lipid rafts, suggesting that the entry of each of these substrates in these particular membrane microdomains is specifically regulated. Our data support the idea that one of the mechanisms regulating ADAM17 substrate cleavage involves protein partitioning in lipid rafts.

  17. Isolation of nano-meso scale detergent resistant membrane that has properties expected of lipid 'rafts'.

    PubMed

    Morris, Roger J; Jen, Angela; Warley, Alice

    2011-03-01

    This review assesses problems that confound attempts to isolate 'raft' domains from cell membranes, focusing in particular upon the isolation of detergent resistant membrane (DRM). Despite its widespread use, this technique is rightly viewed with skepticism by many membrane biochemists and biophysics for reasons that include the inability to isolate DRMs at 37°C, the temperature at which their lipids are supposed to be ordered and so exclude detergents. If solubilization is done in an ionic buffer that preserves the lamellar phase of the metastable inner leaflet lipids, DRMs can readily be isolated at 37°C, and these have many properties expected of lipid rafts. However, to date these DRMs have remained somewhat larger than current concepts of rafts. We describe an adaptation of this method that purifies nano-meso scale DRMs, and could be a significant step towards purifying the membrane of individual 'rafts'. PMID:21214574

  18. Amyloid Oligomer Neurotoxicity, Calcium Dysregulation, and Lipid Rafts

    PubMed Central

    Malchiodi-Albedi, Fiorella; Paradisi, Silvia; Matteucci, Andrea; Frank, Claudio; Diociaiuti, Marco

    2011-01-01

    Amyloid proteins constitute a chemically heterogeneous group of proteins, which share some biophysical and biological characteristics, the principal of which are the high propensity to acquire an incorrect folding and the tendency to aggregate. A number of diseases are associated with misfolding and aggregation of proteins, although only in some of them—most notably Alzheimer's disease (AD) and transmissible spongiform encephalopathies (TSEs)—a pathogenetic link with misfolded proteins is now widely recognized. Lipid rafts (LRs) have been involved in the pathophysiology of diseases associated with protein misfolding at several levels, including aggregation of misfolded proteins, amyloidogenic processing, and neurotoxicity. Among the pathogenic misfolded proteins, the AD-related protein amyloid β (Aβ) is by far the most studied protein, and a large body of evidence has been gathered on the role played by LRs in Aβ pathogenicity. However, significant amount of data has also been collected for several other amyloid proteins, so that their ability to interact with LRs can be considered an additional, shared feature characterizing the amyloid protein family. In this paper, we will review the evidence on the role of LRs in the neurotoxicity of huntingtin, α-synuclein, prion protein, and calcitonin. PMID:21331330

  19. Sphingomyelin distribution in lipid rafts of artificial monolayer membranes visualized by Raman microscopy.

    PubMed

    Ando, Jun; Kinoshita, Masanao; Cui, Jin; Yamakoshi, Hiroyuki; Dodo, Kosuke; Fujita, Katsumasa; Murata, Michio; Sodeoka, Mikiko

    2015-04-14

    Sphingomyelin (SM) and cholesterol (chol)-rich domains in cell membranes, called lipid rafts, are thought to have important biological functions related to membrane signaling and protein trafficking. To visualize the distribution of SM in lipid rafts by means of Raman microscopy, we designed and synthesized an SM analog tagged with a Raman-active diyne moiety (diyne-SM). Diyne-SM showed a strong peak in a Raman silent region that is free of interference from intrinsic vibrational modes of lipids and did not appear to alter the properties of SM-containing monolayers. Therefore, we used Raman microscopy to directly visualize the distribution of diyne-SM in raft-mimicking domains formed in SM/dioleoylphosphatidylcholine/chol ternary monolayers. Raman images visualized a heterogeneous distribution of diyne-SM, which showed marked variation, even within a single ordered domain. Specifically, diyne-SM was enriched in the central area of raft domains compared with the peripheral area. These results seem incompatible with the generally accepted raft model, in which the raft and nonraft phases show a clear biphasic separation. One of the possible reasons is that gradual changes of SM concentration occur between SM-rich and -poor regions to minimize hydrophobic mismatch. We believe that our technique of hyperspectral Raman imaging of a single lipid monolayer opens the door to quantitative analysis of lipid membranes by providing both chemical information and spatial distribution with high (diffraction-limited) spatial resolution. PMID:25825736

  20. Sphingomyelin distribution in lipid rafts of artificial monolayer membranes visualized by Raman microscopy

    PubMed Central

    Ando, Jun; Kinoshita, Masanao; Cui, Jin; Yamakoshi, Hiroyuki; Dodo, Kosuke; Fujita, Katsumasa; Murata, Michio; Sodeoka, Mikiko

    2015-01-01

    Sphingomyelin (SM) and cholesterol (chol)-rich domains in cell membranes, called lipid rafts, are thought to have important biological functions related to membrane signaling and protein trafficking. To visualize the distribution of SM in lipid rafts by means of Raman microscopy, we designed and synthesized an SM analog tagged with a Raman-active diyne moiety (diyne-SM). Diyne-SM showed a strong peak in a Raman silent region that is free of interference from intrinsic vibrational modes of lipids and did not appear to alter the properties of SM-containing monolayers. Therefore, we used Raman microscopy to directly visualize the distribution of diyne-SM in raft-mimicking domains formed in SM/dioleoylphosphatidylcholine/chol ternary monolayers. Raman images visualized a heterogeneous distribution of diyne-SM, which showed marked variation, even within a single ordered domain. Specifically, diyne-SM was enriched in the central area of raft domains compared with the peripheral area. These results seem incompatible with the generally accepted raft model, in which the raft and nonraft phases show a clear biphasic separation. One of the possible reasons is that gradual changes of SM concentration occur between SM-rich and -poor regions to minimize hydrophobic mismatch. We believe that our technique of hyperspectral Raman imaging of a single lipid monolayer opens the door to quantitative analysis of lipid membranes by providing both chemical information and spatial distribution with high (diffraction-limited) spatial resolution. PMID:25825736

  1. Toward atomic force microscopy and mass spectrometry to visualize and identify lipid rafts in plasmodesmata

    PubMed Central

    Naulin, Pamela A.; Alveal, Natalia A.; Barrera, Nelson P.

    2014-01-01

    Plant cell-to-cell communication is mediated by nanopores called plasmodesmata (PDs) which are complex structures comprising plasma membrane (PM), highly packed endoplasmic reticulum and numerous membrane proteins. Although recent advances on proteomics have led to insights into mechanisms of transport, there is still an inadequate characterization of the lipidic composition of the PM where membrane proteins are inserted. It has been postulated that PDs could be formed by lipid rafts, however no structural evidence has shown to visualize and analyse their lipid components. In this perspective article, we discuss proposed experiments to characterize lipid rafts and proteins in the PDs. By using atomic force microscopy (AFM) and mass spectrometry (MS) of purified PD vesicles it is possible to determine the presence of lipid rafts, specific bound proteins and the lipidomic profile of the PD under physiological conditions and after changing transport permeability. In addition, MS can determine the stoichiometry of intact membrane proteins inserted in lipid rafts. This will give novel insights into the role of membrane proteins and lipid rafts on the PD structure. PMID:24910637

  2. Lipid rafts in epithelial brush borders: atypical membrane microdomains with specialized functions.

    PubMed

    Danielsen, E Michael; Hansen, Gert H

    2003-10-31

    Epithelial cells that fulfil high-throughput digestive/absorptive functions, such as small intestinal enterocytes and kidney proximal tubule cells, are endowed with a dense apical brush border. It has long been recognized that the microvillar surface of the brush border is organized in cholesterol/sphingolipid-enriched membrane microdomains commonly known as lipid rafts. More recent studies indicate that microvillar rafts, in particular those of enterocytes, have some unusual properties in comparison with rafts present on the surface of other cell types. Thus, microvillar rafts are stable rather than transient/dynamic, and their core components include glycolipids and the divalent lectin galectin-4, which together can be isolated as "superrafts", i.e., membrane microdomains resisting solubilization with Triton X-100 at physiological temperature. These glycolipid/lectin-based rafts serve as platforms for recruitment of GPI-linked and transmembrane digestive enzymes, most likely as an economizing effort to secure and prolong their digestive capability at the microvillar surface. However, in addition to microvilli, the brush border surface also consists of membrane invaginations between adjacent microvilli, which are the only part of the apical surface sterically accessible for membrane fusion/budding events. Many of these invaginations appear as pleiomorphic, deep apical tubules that extend up to 0.5-1 microm into the underlying terminal web region. Their sensitivity to methyl-beta-cyclodextrin suggests them to contain cholesterol-dependent lipid rafts of a different type from the glycolipid-based rafts at the microvillar surface. The brush border is thus an example of a complex membrane system that harbours at least two different types of lipid raft microdomains, each suited to fulfil specialized functions. This conclusion is in line with an emerging, more varied view of lipid rafts being pluripotent microdomains capable of adapting in size, shape, and content to

  3. Probing Membrane Protein Interactions with Their Lipid Raft Environment Using Single-Molecule Tracking and Bayesian Inference Analysis

    PubMed Central

    Türkcan, Silvan; Richly, Maximilian U.; Alexandrou, Antigoni; Masson, Jean-Baptiste

    2013-01-01

    The statistical properties of membrane protein random walks reveal information on the interactions between the proteins and their environments. These interactions can be included in an overdamped Langevin equation framework where they are injected in either or both the friction field and the potential field. Using a Bayesian inference scheme, both the friction and potential fields acting on the ε-toxin receptor in its lipid raft have been measured. Two types of events were used to probe these interactions. First, active events, the removal of cholesterol and sphingolipid molecules, were used to measure the time evolution of confining potentials and diffusion fields. Second, passive rare events, de-confinement of the receptors from one raft and transition to an adjacent one, were used to measure hopping energies. Lipid interactions with the ε-toxin receptor are found to be an essential source of confinement. ε-toxin receptor confinement is due to both the friction and potential field induced by cholesterol and sphingolipids. Finally, the statistics of hopping energies reveal sub-structures of potentials in the rafts, characterized by small hopping energies, and the difference of solubilization energy between the inner and outer raft area, characterized by higher hopping energies. PMID:23301023

  4. CD82 Restrains Angiogenesis by Altering Lipid Raft Clustering and CD44 Trafficking in Endothelial Cells

    PubMed Central

    Wei, Quan; Zhang, Feng; Richardson, Mekel M.; Roy, Nathan H.; Rodgers, William; Liu, Yuechueng; Zhao, Wenyuan; Fu, Chenying; Ding, Yingjun; Huang, Chao; Chen, Yuanjian; Sun, Yao; Ding, Lexi; Hu, Yang; Ma, Jianxing; Boulton, Michael E.; Pasula, Satish; Wren, Jonathan D.; Tanaka, Satoshi; Huang, Xiaolin; Thali, Markus; Hämmerling, Günter J.; Zhang, Xin A.

    2014-01-01

    Background Angiogenesis is crucial for many pathological processes and becomes a therapeutic strategy against diseases ranging from inflammation to cancer. The regulatory mechanism of angiogenesis remains unclear. Although tetraspanin CD82 is widely expressed in various endothelial cells (ECs), its vascular function is unknown. Methods and Results Angiogenesis was examined in Cd82-null mice with in vivo and ex vivo morphogenesis assays. Cellular functions, molecular interactions, and signaling were analyzed in Cd82-null ECs. Angiogenic responses to various stimuli became markedly increased upon Cd82 ablation. Major changes of Cd82-null ECs were enhanced migration and invasion, likely resulting from the upregulated expression of cell adhesion molecules (CAMs) such as CD44 and integrins at the cell surface and subsequently elevated outside-in signaling. Gangliosides, lipid raft clustering, and CD44-membrane microdomain interactions were increased in the plasma membrane of Cd82-null ECs, leading to less clathrin-independent endocytosis and then more surface presence of CD44. Conclusions Our study reveals that CD82 restrains pathological angiogenesis by inhibiting EC movement, lipid raft clustering and CAM trafficking modulate angiogenic potential, and the perturbation of CD82-ganglioside-CD44 signaling attenuates angiogenesis. PMID:25149363

  5. 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

  6. Proteome scale characterization of human S-acylated proteins in lipid raft-enriched and non-raft membranes.

    PubMed

    Yang, Wei; Di Vizio, Dolores; Kirchner, Marc; Steen, Hanno; Freeman, Michael R

    2010-01-01

    Protein S-acylation (palmitoylation), a reversible post-translational modification, is critically involved in regulating protein subcellular localization, activity, stability, and multimeric complex assembly. However, proteome scale characterization of S-acylation has lagged far behind that of phosphorylation, and global analysis of the localization of S-acylated proteins within different membrane domains has not been reported. Here we describe a novel proteomics approach, designated palmitoyl protein identification and site characterization (PalmPISC), for proteome scale enrichment and characterization of S-acylated proteins extracted from lipid raft-enriched and non-raft membranes. In combination with label-free spectral counting quantitation, PalmPISC led to the identification of 67 known and 331 novel candidate S-acylated proteins as well as the localization of 25 known and 143 novel candidate S-acylation sites. Palmitoyl acyltransferases DHHC5, DHHC6, and DHHC8 appear to be S-acylated on three cysteine residues within a novel CCX(7-13)C(S/T) motif downstream of a conserved Asp-His-His-Cys cysteine-rich domain, which may be a potential mechanism for regulating acyltransferase specificity and/or activity. S-Acylation may tether cytoplasmic acyl-protein thioesterase-1 to membranes, thus facilitating its interaction with and deacylation of membrane-associated S-acylated proteins. Our findings also suggest that certain ribosomal proteins may be targeted to lipid rafts via S-acylation, possibly to facilitate regulation of ribosomal protein activity and/or dynamic synthesis of lipid raft proteins in situ. In addition, bioinformatics analysis suggested that S-acylated proteins are highly enriched within core complexes of caveolae and tetraspanin-enriched microdomains, both cholesterol-rich membrane structures. The PalmPISC approach and the large scale human S-acylated protein data set are expected to provide powerful tools to facilitate our understanding of the

  7. Lipid Rafts Alter the Stability and Activity of the Cholera Toxin A1 Subunit*

    PubMed Central

    Ray, Supriyo; Taylor, Michael; Banerjee, Tuhina; Tatulian, Suren A.; Teter, Ken

    2012-01-01

    Cholera toxin (CT) travels from the cell surface to the endoplasmic reticulum (ER) as an AB holotoxin. ER-specific conditions then promote the dissociation of the catalytic CTA1 subunit from the rest of the toxin. CTA1 is held in a stable conformation by its assembly in the CT holotoxin, but the dissociated CTA1 subunit is an unstable protein that spontaneously assumes a disordered state at physiological temperature. This unfolding event triggers the ER-to-cytosol translocation of CTA1 through the quality control mechanism of ER-associated degradation. The translocated pool of CTA1 must regain a folded, active structure to modify its G protein target which is located in lipid rafts at the cytoplasmic face of the plasma membrane. Here, we report that lipid rafts place disordered CTA1 in a functional conformation. The hydrophobic C-terminal domain of CTA1 is essential for binding to the plasma membrane and lipid rafts. These interactions inhibit the temperature-induced unfolding of CTA1. Moreover, lipid rafts could promote a gain of structure in the disordered, 37 °C conformation of CTA1. This gain of structure corresponded to a gain of function: whereas CTA1 by itself exhibited minimal in vitro activity at 37 °C, exposure to lipid rafts resulted in substantial toxin activity at 37 °C. In vivo, the disruption of lipid rafts with filipin substantially reduced the activity of cytosolic CTA1. Lipid rafts thus exhibit a chaperone-like function that returns disordered CTA1 to an active state and is required for the optimal in vivo activity of CTA1. PMID:22787142

  8. 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. PMID:23665237

  9. 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

  10. 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

    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

  11. Compartmentalization of the exocyst complex in lipid rafts controls Glut4 vesicle tethering.

    PubMed

    Inoue, Mayumi; Chiang, Shian-Huey; Chang, Louise; Chen, Xiao-Wei; Saltiel, Alan R

    2006-05-01

    Lipid raft microdomains act as organizing centers for signal transduction. We report here that the exocyst complex, consisting of Exo70, Sec6, and Sec8, regulates the compartmentalization of Glut4-containing vesicles at lipid raft domains in adipocytes. Exo70 is recruited by the G protein TC10 after activation by insulin and brings with it Sec6 and Sec8. Knockdowns of these proteins block insulin-stimulated glucose uptake. Moreover, their targeting to lipid rafts is required for glucose uptake and Glut4 docking at the plasma membrane. The assembly of this complex also requires the PDZ domain protein SAP97, a member of the MAGUKs family, which binds to Sec8 upon its translocation to the lipid raft. Exocyst assembly at lipid rafts sets up targeting sites for Glut4 vesicles, which transiently associate with these microdomains upon stimulation of cells with insulin. These results suggest that the TC10/exocyst complex/SAP97 axis plays an important role in the tethering of Glut4 vesicles to the plasma membrane in adipocytes. PMID:16525015

  12. Autoimmunoreactive IgGs Against Cardiac Lipid Raft-Associated Proteins in Patients with POTS

    PubMed Central

    Wang, Xiao-Li; Ling, Tian-You; Charlesworth, M. Cristine; Figueroa, Juan J.; Low, Phillip; Shen, Win-Kuang; Lee, Hon-Chi

    2013-01-01

    Lipid rafts are specialized plasma membrane microdomains that serve as platforms for integrating cellular signal transductions. We have recently reported that autoantibodies against cardiac membrane proteins are present in patients with postural orthostatic tachycardia syndrome (POTS). In this study, we examined the presence of autoimmunoreactive IgGs against lipid raft proteins in these patients. IgGs were purified from the sera of 10 patients and 7 normal controls. Cardiac lipid raft preparations were isolated from normal human heart tissue. The lipid raft-associated proteins were resolved by 2DE and immunoblotted against IgGs from each subject. Protein spots that reacted specifically with patient IgGs were identified by nanoLC-MS/MS. Thirty-four such protein spots, and 72 unique proteins were identified. The targets of autoimmunoreactive IgGs include proteins associated with caveolae structure, adrenergic signaling, calcium signaling, cytostructures, chaperone and energy metabolism. Multiple pathways were involved including those that regulate caveolae-mediated signaling, oxidative phosphorylation, fatty acid metabolism, protein ubiquitination, and cardiac β-adrenergic signaling. Our results suggest that cardiac lipid raft-associated proteins are targets of autoimmunoreactive IgGs from patients with POTS. Autoimmunity may play a role in the pathogenesis of POTS. PMID:23562385

  13. Lipid Raft Is Required for PSGL-1 Ligation Induced HL-60 Cell Adhesion on ICAM-1

    PubMed Central

    Xu, Tingshuang; Liu, Wenai; Luo, Jixian; Li, Chunfeng; Ba, Xueqing; Ampah, Khamal Kwesi; Wang, Xiaoguang; Jiang, Yong; Zeng, Xianlu

    2013-01-01

    P-selectin glycoprotein ligand-1 (PSGL-1) and integrins are adhesion molecules that play critical roles in host defense and innate immunity. PSGL-1 mediates leukocyte rolling and primes leukocytes for integrin-mediated adhesion. However, the mechanism that PSGL-1 as a rolling receptor in regulating integrin activation has not been well characterized. Here, we investigate the function of lipid raft in regulating PSGL-1 induced β2 integrin-mediated HL-60 cells adhesion. PSGL-1 ligation with antibody enhances the β2 integrin activation and β2 integrin-dependent adhesion to ICAM-1. Importantly, with the treatment of methyl-β-cyclodextrin (MβCD), we confirm the role of lipid raft in regulating the activation of β2 integrin. Furthermore, we find that the protein level of PSGL-1 decreased in raft fractions in MβCD treated cells. PSGL-1 ligation induces the recruitment of spleen tyrosine kinase (Syk), a tyrosine kinase and Vav1 (the pivotal downstream effector of Syk signaling pathway involved in cytoskeleton regulation) to lipid raft. Inhibition of Syk activity with pharmacologic inhibitor strongly reduces HL-60 cells adhesion, implicating Syk is crucial for PSGL-1 mediated β2 integrin activation. Taken together, we report that ligation of PSGL-1 on HL-60 cells activates β2 integrin, for which lipid raft integrity and Syk activation are responsible. These findings have shed new light on the mechanisms that connect leukocyte initial rolling with subsequent adhesion. PMID:24312591

  14. 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

  15. Molecular targets of (-)-epigallocatechin-3-gallate (EGCG): specificity and interaction with membrane lipid rafts.

    PubMed

    Patra, S K; Rizzi, F; Silva, A; Rugina, D O; Bettuzzi, S

    2008-12-01

    Proteomic studies on anticancer activity of Green Tea Catechins (specifically EGCG) are suggesting a large set of protein targets that may directly interact with EGCG and alter the physiology of diseased cells, including cancer. Of notice, benign cells are usually left untouched. Lipid rafts have been recently recognized as signal processing hubs and suggested to be involved in drug uptake by means of endocytosis. These findings are suggesting new insights on the molecular mechanisms of anticancer drugs action. In the membrane, EGCG is hijacked by the laminin receptor (LamR), a lipid raft protein. Similar to aplidin and edelfosin, EGCG alters membrane domains composition also preventing EGF binding to EGFR, imerization of EGFR and relocation of phosphorylated EGFR to lipid rafts. In vitro studies have recently shown that EGCG also binds both DNA and RNA in GpC-rich regions. This event may importantly affect genes function. Moreover, EGCG was shown to inhibit telomerase, topoisomerase II and DNA methyltransferase 1 (DNMT1), thus ultimately affecting chromatin maintenance and remodeling. But another important alternative pathway besides interaction with specific proteins may play an important role in EGCG action: direct targeting of bioactive membrane platforms, lipid rafts. Structural alteration of the platforms deeply impact (and often inactivates) important pathways involving MAP kinases. The key issue is that, important and specific differences in lipid rafts composition have been found in transformed versus benign cells and apoptotic versus non-apoptotic cells. We suggest here that the anticancer activity of Green Tea Catechins against different kind of cancers may find an explanation in direct targeting of lipid rafts by EGCG. PMID:19261982

  16. Dynamical Clustering and a Mechanism for Raft-like Structures in a Model Lipid Membrane

    PubMed Central

    Starr, Francis W.; Hartmann, Benedikt; Douglas, Jack F.

    2014-01-01

    We use molecular dynamics simulations to examine the dynamical heterogeneity of a model single-component lipid membrane using a coarse-grained representation of lipid molecules. This model qualitatively reproduces the known phase transitions between disordered, ordered, and gel membrane phases, and the phase transitions are accompanied by significant changes in the nature of the lipid dynamics. In particular, lipid diffusion in the liquid-ordered phase is hindered by the transient trapping of molecules by their neighbors, similar to the dynamics of a liquid approaching its glass transition. This transient molecular caging gives rise to two distinct mobility groups within a single-component membrane: lipids that are transiently trapped, and lipids with displacements on the scale of the intermolecular spacing. Most significantly, lipids within these distinct mobility states spatially segregate, creating transient “islands” of enhanced mobility having a size and time scale compatible with lipidrafts,” dynamical structures thought to be important for cell membrane function. Although the dynamic lipid clusters that we observe do not themselves correspond to rafts (which are more complex, multicomponent structures), we hypothesize that such rafts may develop from the same universal mechanism, explaining why raft-like regions should arise, regardless of lipid structural or compositional details. These clusters are strikingly similar to the dynamical clusters found in glass-forming fluids, and distinct from phase-separation clusters. Further examination shows that mobile lipid clusters can be dissected into smaller clusters of cooperatively rearranging molecules. The geometry of these clusters can be understood in the context of branched equilibrium polymers, related to the statistics percolation theory. We discuss how these dynamical structures relate to a range observations on the dynamics of lipid membranes. PMID:24695573

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

    SciTech Connect

    Adebiyi, Adebowale Soni, Hitesh; John, Theresa A.; Yang, Fen

    2014-05-15

    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-1 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.

  18. Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains[S

    PubMed Central

    Betz, Josefine; Bielaszewska, Martina; Thies, Andrea; Humpf, Hans-Ulrich; Dreisewerd, Klaus; Karch, Helge; Kim, Kwang S.; Friedrich, Alexander W.; Müthing, Johannes

    2011-01-01

    Vascular damage caused by Shiga toxin (Stx)-producing Escherichia coli is largely mediated by Stxs, which in particular, injure microvascular endothelial cells in the kidneys and brain. The majority of Stxs preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) and, to a lesser extent, to globotetraosylceramide (Gb4Cer). As clustering of receptor GSLs in lipid rafts is a functional requirement for Stxs, we analyzed the distribution of Gb3Cer and Gb4Cer to membrane microdomains of human brain microvascular endothelial cells (HBMECs) and macrovascular EA.hy 926 endothelial cells by means of anti-Gb3Cer and anti-Gb4Cer antibodies. TLC immunostaining coupled with infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry revealed structural details of various lipoforms of Stx receptors and demonstrated their major distribution in detergent-resistant membranes (DRMs) compared with nonDRM fractions of HBMECs and EA.hy 926 cells. A significant preferential partition of different receptor lipoforms carrying C24:0/C24:1 or C16:0 fatty acid and sphingosine to DRMs was not detected in either cell type. Methyl-β-cyclodextrin (MβCD)-mediated cholesterol depletion resulted in only partial destruction of lipid rafts, accompanied by minor loss of GSLs in HBMECs. In contrast, almost entire disintegration of lipid rafts accompanied by roughly complete loss of GSLs was detected in EA.hy 926 cells after removal of cholesterol, indicating more stable microdomains in HBMECs. Our findings provide first evidence for differently stable microdomains in human endothelial cells from different vascular beds and should serve as the basis for further exploring the functional role of lipid raft-associated Stx receptors in different cell types. PMID:21252262

  19. Endocytosis of Gene Delivery Vectors: From Clathrin-dependent to Lipid Raft-mediated Endocytosis

    PubMed Central

    El-Sayed, Ayman; Harashima, Hideyoshi

    2013-01-01

    The ideal nonviral vector delivers its nucleic acid cargo to a specific intracellular target. Vectors enter cells mainly through endocytosis and are distributed to various intracellular organelles. Recent advances in microscopy, lipidomics, and proteomics confirm that the cell membrane is composed of clusters of lipids, organized in the form of lipid raft domains, together with non-raft domains that comprise a generally disordered lipid milieu. The binding of a nonviral vector to either region can determine the pathway for its endocytic uptake and subsequent intracellular itinerary. Given this model of the cell membrane structure, endocytic pathways should be reclassified in relation to lipid rafts. In this review, we attempt to assess the currently recognized endocytic pathways in mammalian cells. The endocytic pathways are classified in relation to the membrane regions that make up the primary endocytic vesicles. This review covers the well-recognized clathrin-mediated endocytosis (CME), phagocytosis, and macropinocytosis in addition to the less addressed pathways that take place in lipid rafts. These include caveolae-mediated, flotillin-dependent, GTPase regulator associated with focal adhesion kinase-1 (GRAF1)-dependent, adenosine diphosphate-ribosylation factor 6 (Arf6)-dependent, and RhoA-dependent endocytic pathways. We summarize the regulators associated with each uptake pathway and methods for interfering with these regulators are discussed. The fate of endocytic vesicles resulting from each endocytic uptake pathway is highlighted. PMID:23587924

  20. The Lipid Raft-Associated Protein CD98 Is Required for Vaccinia Virus Endocytosis

    PubMed Central

    Schroeder, Nina; Chung, Che-Sheng; Chen, Chein-Hung; Liao, Chung-Lin

    2012-01-01

    Mature vaccinia virus (vaccinia MV) infects a broad range of animals in vivo and cell cultures in vitro; however, the cellular receptors that determine vaccinia MV tropism and entry pathways are poorly characterized. Here, we performed quantitative proteomic analyses of lipid raft-associated proteins upon vaccinia MV entry into HeLa cells. We found that a type II membrane glycoprotein, CD98, is enriched in lipid rafts upon vaccinia MV infection compared to mock-infected HeLa cells. The knockdown of CD98 expression in HeLa cells significantly reduced vaccinia MV entry. Furthermore, CD98 knockout (KO) mouse embryonic fibroblasts (MEFs) also exhibited reduced vaccinia MV infectivity without affecting MV attachment to cells, suggesting a role for CD98 in the postbinding step of virus entry. Further characterization with inhibitors and dominant negative proteins that block different endocytic pathways revealed that vaccinia MV entry into MEFs occurs through a clathrin-independent, caveolin-independent, dynamin-dependent, fluid-phase endocytic pathway, implying that CD98 plays a specific role in the vaccinia MV endocytic pathway. Infections of wild-type and CD98 KO MEF cells with different strains of vaccinia MV provided further evidence that CD98 plays a specific role in MV endocytosis but not in plasma membrane fusion. Finally, different CD98-C69 chimeric proteins were expressed in CD98 KO MEFs, but none were able to reconstitute MV infectivity, suggesting that the overall structure of the CD98 protein is required for vaccinia MV endocytosis. PMID:22345471

  1. Ethanol alters cellular activation and CD14 partitioning in lipid rafts

    SciTech Connect

    Dai Qun; Zhang Jun; Pruett, Stephen B. . E-mail: spruet@lsuhsc.edu

    2005-06-24

    Alcohol consumption interferes with innate immunity. In vivo EtOH administration suppresses cytokine responses induced through Toll-like receptor 4 (TLR4) and inhibits TLR4 signaling. Actually, EtOH exhibits a generalized suppressive effect on signaling and cytokine responses induced by through most TLRs. However, the underlying mechanism remains unknown. RAW264.7 cells were treated with LPS or co-treated with EtOH or with lipid raft-disrupting drugs. TNF-{alpha} production, IRAK-1 activation, and CD14 partition were evaluated. EtOH or nystatin, a lipid raft-disrupting drug, suppressed LPS-induced production of TNF-{alpha}. The suppressive effect of EtOH on LPS-induced TNF-{alpha} production was additive with that of methyl-{beta}-cyclodextrin (MCD), another lipid raft-disrupting drug. EtOH interfered with IRAK-1 activation, an early TLR4 intracellular signaling event. Cell fractionation analyses show that acute EtOH altered LPS-related partition of CD14, a critical component of the LPS receptor complex. These results suggest a novel mechanism of EtOH action that involves interference with lipid raft clustering induced by LPS. This membrane action of EtOH might be one of the mechanisms by which EtOH acts as a generalized suppressor for TLR signaling.

  2. 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

  3. Probing Lipid Membrane Rafts (Microdomains) with Fluorescent Phospholipids

    NASA Astrophysics Data System (ADS)

    Gu, Yongwen; Mitchel, Drake

    2011-10-01

    Membrane rafts are enriched in sphingolipids and cholesterol, they exist in a more ordered state (the liquid-ordered phase; lo) than the bulk membrane (the liquid-disordered phase; ld). Ternary mixtures of palmitoyl-oleoyl-phosphocholine (POPC; 16:0,18:1 PC), sphingomyelin (SPM), and cholesterol (Chol) form membrane rafts over a wide range of molar ratios. We are examining the ability of two fluorescent probes, NBD linked to di-16:0 PE which partitions into the lo phase, and NBD linked to di-18:1 PE which partitions into the ld phase, to detect these two phases. We are also examining the effect of the highly polyunsaturated phospholipid stearoyl-docosahexanoyl-phosphocholine (SDPC; 18:0, 22:6 PC) on the size and stability of POPC/SPM/Chol membrane rafts. We report on the fluorescence lifetime and anisotropy decay dynamics of two fluorescent probes. Data were acquired via frequency-domain measurements from 5 to 250 MHz.

  4. 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

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

    SciTech Connect

    Azouz, Abdallah; Gunn, Teresa M.; Duke-Cohan, Jonathan S. . E-mail: Jonathan_Duke-Cohan@dfci.harvard.edu

    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 of 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.

  6. Lipid Peroxides Promote Large Rafts: Effects of Excitation of Probes in Fluorescence Microscopy and Electrochemical Reactions during Vesicle Formation

    PubMed Central

    Ayuyan, Artem G.; Cohen, Fredric S.

    2006-01-01

    Raft formation and enlargement was investigated in liposomes and supported bilayers prepared from sphingomyelin (SM), cholesterol, and unsaturated phospholipids; NBD-DPPE and rhodamine-(DOPE) were employed as fluorescent probes. Rafts were created by lowering temperature. Maintaining 20 mol % SM, fluorescence microscopy showed that, in the absence of photooxidation, large rafts did not form in giant unilamellar vesicles (GUVs) containing 20 or more mol % cholesterol. But if photooxidation was allowed to proceed, large rafts were readily observed. In population, cuvette experiments, small rafts formed without photooxidation at high cholesterol concentrations. Thus, photooxidation was the cause of raft enlargement during microscopy experiments. Because photooxidation results in peroxidation at lipid double bonds, photosensitization experiments were performed to explicitly produce peroxides of SM and an unsaturated phospholipid. GUVs of high cholesterol content containing the breakdown products of SM-peroxide, but not phospholipid-peroxide, resulted in large rafts after lowering temperature. In addition, GUV production by electroswelling can result in peroxides that cause large raft formation. The use of titanium electrodes eliminates this problem. In conclusion, lipid peroxides and their breakdown products are the cause of large raft formation in GUVs containing biological levels of cholesterol. It is critical that experiments investigating rafts in bilayer membranes avoid the production of peroxides. PMID:16815906

  7. Cholesterol accumulation in Niemann Pick type C (NPC) model cells causes a shift in APP localization to lipid rafts

    SciTech Connect

    Kosicek, Marko; Malnar, Martina; Goate, Alison; Hecimovic, Silva

    2010-03-12

    It has been suggested that cholesterol may modulate amyloid-{beta} (A{beta}) formation, a causative factor of Alzheimer's disease (AD), by regulating distribution of the three key proteins in the pathogenesis of AD ({beta}-amyloid precursor protein (APP), {beta}-secretase (BACE1) and/or presenilin 1 (PS1)) within lipid rafts. In this work we tested whether cholesterol accumulation upon NPC1 dysfunction, which causes Niemann Pick type C disease (NPC), causes increased partitioning of APP into lipid rafts leading to increased CTF/A{beta} formation in these cholesterol-rich membrane microdomains. To test this we used CHO NPC1{sup -/-} cells (NPC cells) and parental CHOwt cells. By sucrose density gradient centrifugation we observed a shift in fl-APP/CTF compartmentalization into lipid raft fractions upon cholesterol accumulation in NPC vs. wt cells. Furthermore, {gamma}-secretase inhibitor treatment significantly increased fl-APP/CTF distribution in raft fractions in NPC vs. wt cells, suggesting that upon cholesterol accumulation in NPC1-null cells increased formation of APP-CTF and its increased processing towards A{beta} occurs in lipid rafts. Our results support that cholesterol overload, such as in NPC disease, leads to increased partitioning of APP/CTF into lipid rafts resulting in increased amyloidogenic processing of APP in these cholesterol-rich membranes. This work adds to the mechanism of the cholesterol-effect on APP processing and the pathogenesis of Alzheimer's disease and supports the role of lipid rafts in these processes.

  8. Epstein-Barr virus LMP1 modulates lipid raft microdomains and the vimentin cytoskeleton for signal transduction and transformation.

    PubMed

    Meckes, David G; Menaker, Nathan F; Raab-Traub, Nancy

    2013-02-01

    The Epstein-Barr virus (EBV) is an important human pathogen that is associated with multiple cancers. The major oncoprotein of the virus, latent membrane protein 1 (LMP1), is essential for EBV B-cell immortalization and is sufficient to transform rodent fibroblasts. This viral transmembrane protein activates multiple cellular signaling pathways by engaging critical effector molecules and thus acts as a ligand-independent growth factor receptor. LMP1 is thought to signal from internal lipid raft containing membranes; however, the mechanisms through which these events occur remain largely unknown. Lipid rafts are microdomains within membranes that are rich in cholesterol and sphingolipids. Lipid rafts act as organization centers for biological processes, including signal transduction, protein trafficking, and pathogen entry and egress. In this study, the recruitment of key signaling components to lipid raft microdomains by LMP1 was analyzed. LMP1 increased the localization of phosphatidylinositol 3-kinase (PI3K) and its activated downstream target, Akt, to lipid rafts. In addition, mass spectrometry analyses identified elevated vimentin in rafts isolated from LMP1 expressing NPC cells. Disruption of lipid rafts through cholesterol depletion inhibited PI3K localization to membranes and decreased both Akt and ERK activation. Reduction of vimentin levels or disruption of its organization also decreased LMP1-mediated Akt and ERK activation and inhibited transformation of rodent fibroblasts. These findings indicate that LMP1 reorganizes membrane and cytoskeleton microdomains to modulate signal transduction. PMID:23152522

  9. Gel-Phase Microdomains and Lipid Rafts in Monolayers Affect the Redox Properties of Ubiquinone-10

    PubMed Central

    Becucci, Lucia; Scaletti, Federica; Guidelli, Rolando

    2011-01-01

    The redox properties of ubiquinone-10 (UQ) were examined in monolayers of mixtures of dioleoylphosphatidylcholine, palmitoylsphingomyelin, and cholesterol of different compositions, self-assembled on a mercury electrode, over the pH range from 7.5 to 9.5. A detailed analysis of the cyclic voltammograms of UQ in the above lipid environments points to a mechanism consisting of an elementary electron transfer step followed by two protonation (or deprotonation) steps in quasiequilibrium and by a further electron transfer step. In a lipid environment of solid-ordered (so) microdomains in a liquid-disordered (ld) matrix, electron transport across the lipid monolayer takes place in the ld phase. In a pure so phase, UQ tends to segregate into UQ-rich pools, exhibiting reversible electron transfer steps. In a lipid environment consisting of liquid-ordered (lo) microdomains (lipid rafts) in an ld matrix, UQ molecules tend to localize along the edge of the lipid rafts. However, in a lipid environment consisting exclusively of lo and so microdomains, UQ molecules tend to segregate into UQ-rich pools. In all lipid environments, electron transport by UQ occurs with the quinone moiety localized on the solution side with respect to the ester linkages of the dioleoylphosphatidylcholine molecules. PMID:21723823

  10. CD82 endocytosis and cholesterol-dependent reorganization of tetraspanin webs and lipid rafts

    PubMed Central

    Xu, Congfeng; Zhang, Yanhui H.; Thangavel, Muthusamy; Richardson, Mekel M.; Liu, Li; Zhou, Bin; Zheng, Yi; Ostrom, Rennolds S.; Zhang, Xin A.

    2009-01-01

    Tetraspanin CD82 suppresses cell migration, tumor invasion, and tumor metastasis. To determine the mechanism by which CD82 inhibits motility, most studies have focused on the cell surface CD82, which forms tetraspanin-enriched microdomains (TEMs) with other transmembrane proteins, such as integrins. In this study, we found that CD82 undergoes endocytosis and traffics to endosomes and lysosomes. To determine the endocytic mechanism of CD82, we demonstrated that dynamin and clathrin are not essential for CD82 internalization. Depletion or sequestration of sterol in the plasma membrane markedly inhibited the endocytosis of CD82. Despite the demand on Cdc42 activity, CD82 endocytosis is distinct from macropinocytosis and the documented dynamin-independent pinocytosis. As a TEM component, CD82 reorganizes TEMs and lipid rafts by redistributing cholesterol into these membrane microdomains. CD82-containing TEMs are characterized by the cholesterol-containing microdomains in the extreme light- and intermediate-density fractions. Moreover, the endocytosis of CD82 appears to alleviate CD82-mediated inhibition of cell migration. Taken together, our studies demonstrate that lipid-dependent endocytosis drives CD82 trafficking to late endosomes and lysosomes, and CD82 reorganizes TEMs and lipid rafts through redistribution of cholesterol.—Xu, C., Zhang, Y. H., Thangavel, M., Richardson, M. M., Liu, L., Zhou, B., Zheng, Y., Ostrom, R. S., Zhang, X. A. CD82 endocytosis and cholesterol-dependent reorganization of tetraspanin webs and lipid rafts. PMID:19497983

  11. Lipid rafts participate in aberrant degradative autophagic-lysosomal pathway of amyloid-beta peptide in Alzheimer's disease

    PubMed Central

    Zhou, Xin; Yang, Chun; Liu, Yufeng; Li, Peng; Yang, Huiying; Dai, Jingxing; Qu, Rongmei; Yuan, Lin

    2014-01-01

    Amyloid-beta peptide is the main component of amyloid plaques, which are found in Alzheimer's disease. The generation and deposition of amyloid-beta is one of the crucial factors for the onset and progression of Alzheimer's disease. Lipid rafts are glycolipid-rich liquid domains of the plasma membrane, where certain types of protein tend to aggregate and intercalate. Lipid rafts are involved in the generation of amyloid-beta oligomers and the formation of amyloid-beta peptides. In this paper, we review the mechanism by which lipid rafts disturb the aberrant degradative autophagic-lysosomal pathway of amyloid-beta, which plays an important role in the pathological process of Alzheimer's disease. Moreover, we describe this mechanism from the view of the Two-system Theory of fasciology and thus, suggest that lipid rafts may be a new target of Alzheimer's disease treatment. PMID:25206748

  12. Complex and Multidimensional Lipid Raft Alterations in a Murine Model of Alzheimer's Disease

    PubMed Central

    Chadwick, Wayne; Brenneman, Randall; Martin, Bronwen; Maudsley, Stuart

    2010-01-01

    Various animal models of Alzheimer's disease (AD) have been created to assist our appreciation of AD pathophysiology, as well as aid development of novel therapeutic strategies. Despite the discovery of mutated proteins that predict the development of AD, there are likely to be many other proteins also involved in this disorder. Complex physiological processes are mediated by coherent interactions of clusters of functionally related proteins. Synaptic dysfunction is one of the hallmarks of AD. Synaptic proteins are organized into multiprotein complexes in high-density membrane structures, known as lipid rafts. These microdomains enable coherent clustering of synergistic signaling proteins. We have used mass analytical techniques and multiple bioinformatic approaches to better appreciate the intricate interactions of these multifunctional proteins in the 3xTgAD murine model of AD. Our results show that there are significant alterations in numerous receptor/cell signaling proteins in cortical lipid rafts isolated from 3xTgAD mice. PMID:21151659

  13. Disruption of Lipid Rafts Interferes with the Interaction of Toxoplasma gondii with Macrophages and Epithelial Cells

    PubMed Central

    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 (MβCD) 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. PMID:24734239

  14. HIV-1 Vpu's lipid raft association is dispensable for counteraction of the particle release restriction imposed by CD317/Tetherin

    SciTech Connect

    Fritz, Joeelle V. Tibroni, Nadine Keppler, Oliver T. Fackler, Oliver T.

    2012-03-01

    HIV-1 Vpu antagonizes the block to particle release mediated by CD317 (BST-2/HM1.24/Tetherin) via incompletely understood mechanisms. Vpu and CD317 partially reside in cholesterol-rich lipid rafts where HIV-1 budding preferentially occurs. Here we find that lipid raft association of ectopically expressed or endogenous CD317 was unaltered upon co-expression with Vpu or following HIV-1 infection. Similarly, Vpu's lipid raft association remained unchanged upon expression of CD317. We identify amino acids V25 and Y29 of Vpu as crucial for microdomain partitioning and single substitution of these amino acids resulted in Vpu variants with markedly reduced or undetectable lipid raft association. These mutations did not affect Vpu's subcellular distribution and binding capacity to CD317, nor its ability to downmodulate cell surface CD317 and promote HIV-1 release from CD317-positive cells. We conclude that (i) lipid raft incorporation is dispensable for Vpu-mediated CD317 antagonism and (ii) Vpu does not antagonize CD317 by extraction from lipid rafts.

  15. Alterations in cholesterol and ganglioside GM1 content of lipid rafts in platelets from patients with Alzheimer disease.

    PubMed

    Liu, Li; Zhang, Ke; Tan, Liang; Chen, Yu-Hua; Cao, Yun-Peng

    2015-01-01

    The aim of this study was to investigate the changes in the protein, cholesterol, and ganglioside GM1 content of lipid rafts in platelets from patients with Alzheimer disease (AD), and identify potential blood biomarkers of the disease. A total of 31 Chinese patients with AD and 31 aged-matched control subjects were selected. Lipid rafts were isolated from platelets using Optiprep gradient centrifugation. The protein content of lipid rafts was evaluated using Micro BCA assay, the cholesterol content using molecular probes, ganglioside GM1 content using colorimetry and dot-blotting analysis. The results showed that the cholesterol and ganglioside GM1 content of lipid rafts from platelets was significantly higher in patients with AD than aged-matched control subjects, whereas the protein content of lipid rafts did not show any differences between the 2 groups. These results indicate that the increases in the cholesterol and ganglioside GM1 content of lipid rafts from the platelets of patients with AD might serve as a biochemical adjunct to the clinical diagnosis of AD. PMID:24759545

  16. Primary Human Leukocyte Subsets Differentially Express Vaccinia Virus Receptors Enriched in Lipid Rafts

    PubMed Central

    Byrd, Daniel; Amet, Tohti; Hu, Ningjie; Lan, Jie; Hu, Sishun

    2013-01-01

    Poxviruses, including vaccinia virus (VV) and canarypox virus (ALVAC), do not indiscriminately infect all cell types of the primary human leukocytes (PHLs) that they encounter but instead demonstrate an extremely strong bias toward infection of monocytes and monocyte lineage cells. We studied the specific molecular events that determine the VV tropism for major PHL subsets including monocytes, B cells, neutrophils, NK cells, and T cells. We found that VV exhibited an extremely strong bias of cell surface protein-dependent binding to monocytes, B cells, and activated T cells to a similar degree and to neutrophils to a much lesser extent. Resting T cells and resting NK cells exhibited only trace amounts of VV binding. Activated T cells, however, became permissive to VV binding, infection, and replication, while activated NK cells still resisted VV binding. VV binding strongly colocalized with lipid rafts on the surfaces of all VV binding-susceptible PHL subsets, even when lipid rafts were relocated to cell uropods upon cell polarization. Immunosera raised against detergent-resistant membranes (DRMs) from monocytes or activated T cells, but not resting T cells, effectively cross-blocked VV binding to and infection of PHL subsets. CD29 and CD98, two lipid raft-associated membrane proteins that had been found to be important for VV entry into HeLa cells, had no effect on VV binding to and infection of primary activated T cells. Our data indicate that PHL subsets express VV protein receptors enriched in lipid rafts and that receptors are cross-presented on all susceptible PHLs. PMID:23785200

  17. Further evidence that paroxysmal nocturnal haemoglobinuria is a disorder of defective cell membrane lipid rafts

    PubMed Central

    Ratajczak, Mariusz Z; Borkowska, Sylwia; Mierzejewska, Kasia; Kucia, Magda; Mendek-Czajkowska, Ewa; Suszynska, Malwina; Sharma, Vivek A; Deptala, Andrzej; Song, Wechao; Platzbecker, Uwe; Larratt, Loree; Janowska-Wieczorek, Anna; Maciejewski, Jarek; Ratajczak, Janina

    2015-01-01

    The glycolipid glycosylphosphatidylinositol anchor (GPI-A) plays an important role in lipid raft formation, which is required for proper expression on the cell surface of two inhibitors of the complement cascade, CD55 and CD59. The absence of these markers from the surface of blood cells, including erythrocytes, makes the cells susceptible to complement lysis, as seen in patients suffering from paroxysmal nocturnal haemoglobinuria (PNH). However, the explanation for why PNH-affected hematopoietic stem/progenitor cells (HSPCs) expand over time in BM is still unclear. Here, we propose an explanation for this phenomenon and provide evidence that a defect in lipid raft formation in HSPCs leads to defective CXCR4- and VLA-4-mediated retention of these cells in BM. In support of this possibility, BM-isolated CD34+ cells from PNH patients show a defect in the incorporation of CXCR4 and VLA-4 into membrane lipid rafts, respond weakly to SDF-1 stimulation, and show defective adhesion to fibronectin. Similar data were obtained with the GPI-A− Jurkat cell line. Moreover, we also report that chimeric mice transplanted with CD55−/− CD59−/− BM cells but with proper GPI-A expression do not expand over time in transplanted hosts. On the basis of these findings, we propose that a defect in lipid raft formation in PNH-mutated HSPCs makes these cells more mobile, so that they expand and out-compete normal HSPCs from their BM niches over time. PMID:26033571

  18. HTLV-1 Tax deregulates autophagy by recruiting autophagic molecules into lipid raft microdomains

    PubMed Central

    Ren, Tong; Takahashi, Yoshinori; Liu, Xin; Loughran, Thomas P.; Sun, Shao-Cong; Wang, Hong-Gang; Cheng, Hua

    2014-01-01

    The retroviral oncoprotein Tax from Human T cell leukemia virus type 1 (HTLV-1), an etiological factor that causes adult T cell leukemia and lymphoma, plays a crucial role in initiating T lymphocyte transformation by inducing oncogenic signaling activation. We here report that Tax is a determining factor for dysregulation of autophagy in HTLV-1-transformed T cells and Tax-immortalized CD4 memory T cells. Tax facilitated autophagic process by activating IκB kinase complex, which subsequently recruited an autophagy molecular complex containing Beclin1 and Bif-1 to the lipid raft microdomains. Tax engaged a crosstalk between IκB kinase complex and autophagic molecule complex by directly interacting with both complexes, promoting assembly of LC3+ autophagosomes. Moreover, expression of lipid raft-targeted Bif-1 or Beclin1 was sufficient to induce formation of LC3+ autophagosomes, suggesting that Tax recruitment of autophagic molecules to lipid rafts is a dominant strategy to deregulate autophagy in the context of HTLV-1 transformation of T cells. Furthermore, depletion of autophagy molecules such as Beclin1 and PI3 kinase class III resulted in impaired growth of HTLV-1-transformed T cells, indicating a critical role of Tax-deregulated autophagy in promoting survival and transformation of virally infected T cells. PMID:24362528

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

    SciTech Connect

    Heberle, Frederick A; Doktorova, Milka; Goh, Shih Lin; Standaert, Robert F; Katsaras, John; Feigenson, Gerald

    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 techniques 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.

  20. Hybrid and nonhybrid lipids exert common effects on membrane raft size and morphology.

    PubMed

    Heberle, Frederick A; Doktorova, Milka; Goh, Shih Lin; Standaert, Robert F; Katsaras, John; Feigenson, Gerald W

    2013-10-01

    Nanometer-scale domains in cholesterol-rich 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 chain-asymmetric 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 techniques 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. PMID:24041024

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

    PubMed

    Marquardt, Drew; Heberle, Frederick A; Nickels, Jonathan D; Pabst, Georg; Katsaras, John

    2015-12-21

    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. 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. PMID:26428538

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

    PubMed Central

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

    2015-01-01

    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. 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. PMID:26428538

  3. Lipid rafts, KCa/ClCa/Ca2+ channel complexes and EGFR signaling: Novel targets to reduce tumor development by lipids?

    PubMed

    Guéguinou, Maxime; Gambade, Audrey; Félix, Romain; Chantôme, Aurélie; Fourbon, Yann; Bougnoux, Philippe; Weber, Günther; Potier-Cartereau, Marie; Vandier, Christophe

    2015-10-01

    Membrane lipid rafts are distinct plasma membrane nanodomains that are enriched with cholesterol, sphingolipids and gangliosides, with occasional presence of saturated fatty acids and phospholipids containing saturated acyl chains. It is well known that they organize receptors (such as Epithelial Growth Factor Receptor), ion channels and their downstream acting molecules to regulate intracellular signaling pathways. Among them are Ca2+ signaling pathways, which are modified in tumor cells and inhibited upon membrane raft disruption. In addition to protein components, lipids from rafts also contribute to the organization and function of Ca2+ signaling microdomains. This article aims to focus on the lipid raft KCa/ClCa/Ca2+ channel complexes that regulate Ca2+ and EGFR signaling in cancer cells, and discusses the potential modification of these complexes by lipids as a novel therapeutic approach in tumor development. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers. PMID:25450343

  4. Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking.

    PubMed

    Wu, Hsiao-Mei; Lin, Ying-Hsiu; Yen, Tzu-Chi; Hsieh, Chia-Lung

    2016-01-01

    Lipid rafts are membrane nanodomains that facilitate important cell functions. Despite recent advances in identifying the biological significance of rafts, nature and regulation mechanism of rafts are largely unknown due to the difficulty of resolving dynamic molecular interaction of rafts at the nanoscale. Here, we investigate organization and single-molecule dynamics of rafts by monitoring lateral diffusion of single molecules in raft-containing reconstituted membranes supported on mica substrates. Using high-speed interferometric scattering (iSCAT) optical microscopy and small gold nanoparticles as labels, motion of single lipids is recorded via single-particle tracking (SPT) with nanometer spatial precision and microsecond temporal resolution. Processes of single molecules partitioning into and escaping from the raft-mimetic liquid-ordered (Lo) domains are directly visualized in a continuous manner with unprecedented clarity. Importantly, we observe subdiffusion of saturated lipids in the Lo domain in microsecond timescale, indicating the nanoscopic heterogeneous molecular arrangement of the Lo domain. Further analysis of the diffusion trajectory shows the presence of nano-subdomains of the Lo phase, as small as 10 nm, which transiently trap the lipids. Our results provide the first experimental evidence of non-uniform molecular organization of the Lo phase, giving a new view of how rafts recruit and confine molecules in cell membranes. PMID:26861908

  5. Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking

    PubMed Central

    Wu, Hsiao-Mei; Lin, Ying-Hsiu; Yen, Tzu-Chi; Hsieh, Chia-Lung

    2016-01-01

    Lipid rafts are membrane nanodomains that facilitate important cell functions. Despite recent advances in identifying the biological significance of rafts, nature and regulation mechanism of rafts are largely unknown due to the difficulty of resolving dynamic molecular interaction of rafts at the nanoscale. Here, we investigate organization and single-molecule dynamics of rafts by monitoring lateral diffusion of single molecules in raft-containing reconstituted membranes supported on mica substrates. Using high-speed interferometric scattering (iSCAT) optical microscopy and small gold nanoparticles as labels, motion of single lipids is recorded via single-particle tracking (SPT) with nanometer spatial precision and microsecond temporal resolution. Processes of single molecules partitioning into and escaping from the raft-mimetic liquid-ordered (Lo) domains are directly visualized in a continuous manner with unprecedented clarity. Importantly, we observe subdiffusion of saturated lipids in the Lo domain in microsecond timescale, indicating the nanoscopic heterogeneous molecular arrangement of the Lo domain. Further analysis of the diffusion trajectory shows the presence of nano-subdomains of the Lo phase, as small as 10 nm, which transiently trap the lipids. Our results provide the first experimental evidence of non-uniform molecular organization of the Lo phase, giving a new view of how rafts recruit and confine molecules in cell membranes. PMID:26861908

  6. Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking

    NASA Astrophysics Data System (ADS)

    Wu, Hsiao-Mei; Lin, Ying-Hsiu; Yen, Tzu-Chi; Hsieh, Chia-Lung

    2016-02-01

    Lipid rafts are membrane nanodomains that facilitate important cell functions. Despite recent advances in identifying the biological significance of rafts, nature and regulation mechanism of rafts are largely unknown due to the difficulty of resolving dynamic molecular interaction of rafts at the nanoscale. Here, we investigate organization and single-molecule dynamics of rafts by monitoring lateral diffusion of single molecules in raft-containing reconstituted membranes supported on mica substrates. Using high-speed interferometric scattering (iSCAT) optical microscopy and small gold nanoparticles as labels, motion of single lipids is recorded via single-particle tracking (SPT) with nanometer spatial precision and microsecond temporal resolution. Processes of single molecules partitioning into and escaping from the raft-mimetic liquid-ordered (Lo) domains are directly visualized in a continuous manner with unprecedented clarity. Importantly, we observe subdiffusion of saturated lipids in the Lo domain in microsecond timescale, indicating the nanoscopic heterogeneous molecular arrangement of the Lo domain. Further analysis of the diffusion trajectory shows the presence of nano-subdomains of the Lo phase, as small as 10 nm, which transiently trap the lipids. Our results provide the first experimental evidence of non-uniform molecular organization of the Lo phase, giving a new view of how rafts recruit and confine molecules in cell membranes.

  7. Sphingomyelin/Phosphatidylcholine/Cholesterol Phase Diagram: Boundaries and Composition of Lipid Rafts

    PubMed Central

    de Almeida, Rodrigo F. M.; Fedorov, Aleksandre; Prieto, Manuel

    2003-01-01

    The ternary system palmitoylsphingomyelin (PSM)/palmitoyloleoylphosphatidylcholine (POPC)/cholesterol is used to model lipid rafts. The phase behavior of the three binary systems PSM/POPC, PSM/cholesterol, and POPC/cholesterol is first experimentally determined. Phase coexistence boundaries are then determined for ternary mixtures at room temperature (23°C) and the ternary phase diagram at that temperature is obtained. From the diagram at 23°C and the binary phase diagrams, a reasonable expectation is drawn for the ternary phase diagram at 37°C. Several photophysical methodologies are employed that do not involve detergent extraction, in addition to literature data (e.g., differential scanning calorimetry) and thermodynamic rules. For the ternary phase diagrams, some tie-lines are calculated, including the one that contains the PSM/POPC/ cholesterol 1:1:1 mixture, which is often used in model raft studies. The diagrams here described are used to rationalize literature results, some of them apparently discrepant, and to discuss lipid rafts within the framework of liquid-ordered/liquid-disordered phase coexistence. PMID:14507704

  8. 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. PMID:26507543

  9. Ligand Binding Alters Dimerization and Sequestering of Urokinase Receptors in Raft-Mimicking Lipid Mixtures

    PubMed Central

    Ge, Yifan; Siegel, Amanda P.; Jordan, Rainer; Naumann, Christoph A.

    2014-01-01

    Lipid heterogeneities, such as lipid rafts, are widely considered to be important for the sequestering of membrane proteins in plasma membranes, thereby influencing membrane protein functionality. However, the underlying mechanisms of such sequestration processes remain elusive, in part, due to the small size and often transient nature of these functional membrane heterogeneities in cellular membranes. To overcome these challenges, here we report the sequestration behavior of urokinase receptor (uPAR), a glycosylphosphatidylinositol-anchored protein, in a planar model membrane platform with raft-mimicking lipid mixtures of well-defined compositions using a powerful optical imaging platform consisting of confocal spectroscopy XY-scans, photon counting histogram, and fluorescence correlation spectroscopy analyses. This methodology provides parallel information about receptor sequestration, oligomerization state, and lateral mobility with single molecule sensitivity. Most notably, our experiments demonstrate that moderate changes in uPAR sequestration are not only associated with modifications in uPAR dimerization levels, but may also be linked to ligand-mediated allosteric changes of these membrane receptors. Our data show that these modifications in uPAR sequestration can be induced by exposure to specific ligands (urokinase plasminogen activator, vitronectin), but not via adjustment of the cholesterol level in the planar model membrane system. Good agreement of our key findings with published results on cell membranes confirms the validity of our model membrane approach. We hypothesize that the observed mechanism of receptor translocation in the presence of raft-mimicking lipid mixtures is also applicable to other glycosylphosphatidylinositol-anchored proteins. PMID:25418095

  10. 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

  11. Alterations in lipid raft composition and dynamics contribute to abnormal T cell responses in systemic lupus erythematosus.

    PubMed

    Krishnan, Sandeep; Nambiar, Madhusoodana P; Warke, Vishal G; Fisher, Carolyn U; Mitchell, Jeanne; Delaney, Nancy; Tsokos, George C

    2004-06-15

    In response to appropriate stimulation, T lymphocytes from systemic lupus erythematosus (SLE) patients exhibit increased and faster intracellular tyrosine phosphorylation and free calcium responses. We have explored whether the composition and dynamics of lipid rafts are responsible for the abnormal T cell responses in SLE. SLE T cells generate and possess higher amounts of ganglioside-containing lipid rafts and, unlike normal T cells, SLE T cell lipid rafts include FcRgamma and activated Syk kinase. IgM anti-CD3 Ab-mediated capping of TCR complexes occurs more rapidly in SLE T cells and concomitant with dramatic acceleration of actin polymerization kinetics. The significance of these findings is evident from the observation that cross-linking of lipid rafts evokes earlier and higher calcium responses in SLE T cells. Thus, we propose that alterations in the lipid raft signaling machinery represent an important mechanism that is responsible for the heightened and accelerated T cell responses in SLE. PMID:15187166

  12. LIPID RAFTS, FLUID/FLUID PHASE SEPARATION, AND THEIR RELEVANCE TO PLASMA MEMBRANE STRUCTURE AND FUNCTION

    PubMed Central

    Sengupta, Prabuddha; Baird, Barbara; Holowka, David

    2007-01-01

    Novel biophysical approaches combined with modeling and new biochemical data have helped to recharge the lipid raft field and have contributed to the generation of a refined model of plasma membrane organization. In this review, we summarize new information in the context of previous literature to provide new insights into the spatial organization and dynamics of lipids and proteins in the plasma membrane of live cells. Recent findings of large-scale separation of liquid-ordered and liquid-disordered phases in plasma membrane vesicles demonstrate this capacity within the complex milieu of plasma membrane proteins and lipids. Roles for membrane heterogeneity and reorganization in immune cell activation are discussed in light of this new information. PMID:17764993

  13. Bordetella parapertussis Survives inside Human Macrophages in Lipid Raft-Enriched Phagosomes

    PubMed Central

    Gorgojo, Juan; Harvill, Eric T.

    2014-01-01

    Bordetella parapertussis is a human pathogen that causes whooping cough. The increasing incidence of B. parapertussis has been attributed to the lack of cross protection induced by pertussis vaccines. It was previously shown that B. parapertussis is able to avoid bacterial killing by polymorphonuclear leukocytes (PMN) if specific opsonic antibodies are not present at the site of interaction. Here, we evaluated the outcome of B. parapertussis innate interaction with human macrophages, a less aggressive type of cell and a known reservoir of many persistent pathogens. The results showed that in the absence of opsonins, O antigen allows B. parapertussis to inhibit phagolysosomal fusion and to remain alive inside macrophages. The O antigen targets B. parapertussis to lipid rafts that are retained in the membrane of phagosomes that do not undergo lysosomal maturation. Forty-eight hours after infection, wild-type B. parapertussis bacteria but not the O antigen-deficient mutants were found colocalizing with lipid rafts and alive in nonacidic compartments. Taken together, our data suggest that in the absence of opsonic antibodies, B. parapertussis survives inside macrophages by preventing phagolysosomal maturation in a lipid raft- and O antigen-dependent manner. Two days after infection, about 15% of macrophages were found loaded with live bacteria inside flotillin-enriched phagosomes that had access to nutrients provided by the host cell recycling pathway, suggesting the development of an intracellular infection. IgG opsonization drastically changed this interaction, inducing efficient bacterial killing. These results highlight the need for B. parapertussis opsonic antibodies to induce bacterial clearance and prevent the eventual establishment of cellular reservoirs of this pathogen. PMID:25267839

  14. 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

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

    SciTech Connect

    Mulari, Mika T.K. Nars, Martin; Laitala-Leinonen, Tiina; Kaisto, Tuula; Metsikkoe, Kalervo; Sun Yi; Vaeaenaenen, H. Kalervo

    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 FSD 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.

  16. Alemtuzumab induces caspase-independent cell death in human chronic lymphocytic leukemia cells through a lipid raft-dependent mechanism.

    PubMed

    Mone, A P; Cheney, C; Banks, A L; Tridandapani, S; Mehter, N; Guster, S; Lin, T; Eisenbeis, C F; Young, D C; Byrd, J C

    2006-02-01

    Alemtuzumab is a humanized IgG1 kappa antibody directed against CD52, a glycosyl-phosphatidylinositol linked cell-membrane protein of unknown function. Herein, we demonstrate that alemtuzumab promotes rapid death of chronic lymphocytic leukemia (CLL) cells in vitro, in a complement and accessory cell free system. Using minimal detergent solubilization of CLL membranes, we found that CD52 colocalizes with ganglioside GM-1, a marker of membrane rafts. Fluorescence microscopy revealed that upon crosslinking CD52 with alemtuzumab+anti-Fc IgG, large patches, and in many cases caps, enriched in CD52 and GM-1 formed upon the CLL cell plasma membrane. Depletion of membrane cholesterol or inhibition of actin polymerization significantly diminished the formation of alemtuzumab-induced caps and reduced alemtuzumab-mediated CLL cell death. We compared alemtuzumab-induced direct cytotoxicity, effector cell-mediated toxicity and complement-mediated cytotoxicity of CLL cells to normal T cells. The direct cytotoxicity and observed capping was significantly greater for CLL cells as compared to normal T cells. Cell-mediated and complement-mediated cytotoxicity did not significantly differ between the two cell types. In summary, our data support the hypothesis that alemtuzumab can initiate CLL cell death by crosslinking CD52-enriched lipid rafts. Furthermore, the differential direct cytotoxic effect suggests that CD52 directed antibodies could possibly be engineered to more specifically target CLL cells. PMID:16341049

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

    PubMed

    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

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

    DOE PAGESBeta

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

    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

  19. 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

  20. Electrophilic nitro-fatty acids inhibit vascular inflammation by disrupting LPS-dependent TLR4 signalling in lipid rafts

    PubMed Central

    Villacorta, Luis; Chang, Lin; Salvatore, Sonia R.; Ichikawa, Tomonaga; Zhang, Jifeng; Petrovic-Djergovic, Danica; Jia, Lingyun; Carlsen, Harald; Schopfer, Francisco J.; Freeman, Bruce A.; Chen, Y. Eugene

    2013-01-01

    Aims Electrophilic fatty acid nitroalkene derivatives, products of unsaturated fatty acid nitration, exert long-term cardiovascular protection in experimental models of metabolic and cardiovascular diseases. The goal of this study is to examine the effects of nitro-fatty acids in the regulation of upstream signalling events in nuclear factor-κB (NF-κB) activation and determine whether low-dose acute administration of nitro-fatty acids reduces vascular inflammation in vivo. Methods and results Using NF-κB-luciferase transgenic mice, it was determined that pre-emptive treatment with nitro-oleic acid (OA-NO2), but not oleic acid (OA) inhibits lipopolysaccharide (LPS)-induced NF-κB activation both in vivo and in isolated macrophages. Acute intravenous administration of OA-NO2 was equally effective to inhibit leukocyte recruitment to the vascular endothelium assessed by intravital microscopy and significantly reduces aortic expression of adhesion molecules. An acute treatment with OA-NO2 in vivo yielding nanomolar concentrations in plasma, is sufficient to inhibit LPS-induced Toll-like receptor 4 (TLR4)-induced cell surface expression in leukocytes and NF-κB activation. In vitro experiments reveal that OA-NO2 suppresses LPS-induced TLR4 signalling, inhibitor of κB (IκBα) phosphorylation and ubiquitination, phosphorylation of the IκB kinase (IKK), impairing the recruitment of the TLR4 and TNF receptor associated factor 6 (TRAF6) to the lipid rafts compartments. Conclusion These studies demonstrate that acute administration of nitro-fatty acids is effective to reduce vascular inflammation in vivo. These findings reveal a direct role of nitro-fatty acids in the disruption of the TLR4 signalling complex in lipid rafts, upstream events of the NF-κB pathway, leading to resolution of pro-inflammatory activation of NF-κB in the vasculature. PMID:23334216

  1. Uptake of granulysin via lipid rafts leads to lysis of intracellular Listeria innocua.

    PubMed

    Walch, Michael; Eppler, Elisabeth; Dumrese, Claudia; Barman, Hanna; Groscurth, Peter; Ziegler, Urs

    2005-04-01

    The bacteriolytic activity of CTL is mediated by granulysin, which has been reported to kill intracellular Mycobacterium tuberculosis in dendritic cells (DC) with high efficiency. Despite that crucial effector function, the killing mechanism and uptake of granulysin into target cells have not been well investigated. To this end we analyzed granulysin binding, uptake, and the subsequent lysis of intracellular Listeria innocua in human DC. Recombinant granulysin was found to be actively taken up by DC into early endosomal Ag 1-labeled endosomes, as detected by immunofluorescence. Further transfer to L. innocua-containing phagosomes was indicated by colocalization of bacterial DNA with granulysin. After uptake of granulysin by DC, lysis of L. innocua was found in a dose-dependent manner. Uptake as well as lysis of Listeria were inhibited after blocking endocytosis by lowering the temperature and by cholesterol depletion of DC. Colocalization of granulysin with cholera toxin during uptake showed binding to and internalization via lipid rafts. In contrast to cholera toxin, which was targeted to the perinuclear compartment, granulysin was found exclusively in endosomal-phagosomal vesicles. Lipid raft microdomains, enriched in the immunological synapse, may thus enhance uptake and transfer of granulysin into bacterial infected host cells. PMID:15778384

  2. Phosphatidylinositol 4-Phosphate 5-Kinase β Controls Recruitment of Lipid Rafts into the Immunological Synapse.

    PubMed

    Kallikourdis, Marinos; Trovato, Anna Elisa; Roselli, Giuliana; Muscolini, Michela; Porciello, Nicla; Tuosto, Loretta; Viola, Antonella

    2016-02-15

    Phosphatidylinositol 4,5-biphosphate (PIP2) is critical for T lymphocyte activation serving as a substrate for the generation of second messengers and the remodeling of actin cytoskeleton necessary for the clustering of lipid rafts, TCR, and costimulatory receptors toward the T:APC interface. Spatiotemporal analysis of PIP2 synthesis in T lymphocytes suggested that distinct isoforms of the main PIP2-generating enzyme, phosphatidylinositol 4-phosphate 5-kinase (PIP5K), play a differential role on the basis of their distinct localization. In this study, we analyze the contribution of PIP5Kβ to T cell activation and show that CD28 induces the recruitment of PIP5Kβ to the immunological synapse, where it regulates filamin A and lipid raft accumulation, as well as T cell activation, in a nonredundant manner. Finally, we found that Vav1 and the C-terminal 83 aa of PIP5Kβ are pivotal for the PIP5Kβ regulatory functions in response to CD28 stimulation. PMID:26773155

  3. Saltatory conduction in unmyelinated axons: clustering of Na+ channels on lipid rafts enables micro-saltatory conduction in C-fibers

    PubMed Central

    Neishabouri, Ali; Faisal, A. Aldo

    2014-01-01

    The action potential (AP), the fundamental signal of the nervous system, is carried by two types of axons: unmyelinated and myelinated fibers. In the former the action potential propagates continuously along the axon as established in large-diameter fibers. In the latter axons the AP jumps along the nodes of Ranvier—discrete, anatomically specialized regions which contain very high densities of sodium ion (Na+) channels. Therefore, saltatory conduction is thought as the hallmark of myelinated axons, which enables faster and more reliable propagation of signals than in unmyelinated axons of same outer diameter. Recent molecular anatomy showed that in C-fibers, the very thin (0.1 μm diameter) axons of the peripheral nervous system, Nav1.8 channels are clustered together on lipid rafts that float in the cell membrane. This localized concentration of Na+ channels resembles in structure the ion channel organization at the nodes of Ranvier, yet it is currently unknown whether this translates into an equivalent phenomenon of saltatory conduction or related-functional benefits and efficiencies. Therefore, we modeled biophysically realistic unmyelinated axons with both conventional and lipid-raft based organization of Na+ channels. We find that APs are reliably conducted in a micro-saltatory fashion along lipid rafts. Comparing APs in unmyelinated fibers with and without lipid rafts did not reveal any significant difference in either the metabolic cost or AP propagation velocity. By investigating the efficiency of AP propagation over Nav1.8 channels, we find however that the specific inactivation properties of these channels significantly increase the metabolic cost of signaling in C-fibers. PMID:25352785

  4. Saltatory conduction in unmyelinated axons: clustering of Na(+) channels on lipid rafts enables micro-saltatory conduction in C-fibers.

    PubMed

    Neishabouri, Ali; Faisal, A Aldo

    2014-01-01

    THE ACTION POTENTIAL (AP), THE FUNDAMENTAL SIGNAL OF THE NERVOUS SYSTEM, IS CARRIED BY TWO TYPES OF AXONS: unmyelinated and myelinated fibers. In the former the action potential propagates continuously along the axon as established in large-diameter fibers. In the latter axons the AP jumps along the nodes of Ranvier-discrete, anatomically specialized regions which contain very high densities of sodium ion (Na(+)) channels. Therefore, saltatory conduction is thought as the hallmark of myelinated axons, which enables faster and more reliable propagation of signals than in unmyelinated axons of same outer diameter. Recent molecular anatomy showed that in C-fibers, the very thin (0.1 μm diameter) axons of the peripheral nervous system, Nav1.8 channels are clustered together on lipid rafts that float in the cell membrane. This localized concentration of Na(+) channels resembles in structure the ion channel organization at the nodes of Ranvier, yet it is currently unknown whether this translates into an equivalent phenomenon of saltatory conduction or related-functional benefits and efficiencies. Therefore, we modeled biophysically realistic unmyelinated axons with both conventional and lipid-raft based organization of Na(+) channels. We find that APs are reliably conducted in a micro-saltatory fashion along lipid rafts. Comparing APs in unmyelinated fibers with and without lipid rafts did not reveal any significant difference in either the metabolic cost or AP propagation velocity. By investigating the efficiency of AP propagation over Nav1.8 channels, we find however that the specific inactivation properties of these channels significantly increase the metabolic cost of signaling in C-fibers. PMID:25352785

  5. Deep-apical tubules: dynamic lipid-raft microdomains in the brush-border region of enterocytes.

    PubMed

    Hansen, Gert H; Pedersen, Jens; Niels-Christiansen, Lise-Lotte; Immerdal, Lissi; Danielsen, E Michael

    2003-07-01

    The brush border of small intestinal enterocytes is highly enriched in cholesterol- and glycosphingolipid-containing membrane microdomains, commonly termed as lipid 'rafts'. Functionally, transcytosis of IgA and exocytosis of newly made brush-border proteins in enterocytes occur through apical lipid raft-containing compartments, but little is otherwise known about these raft microdomains. We therefore studied in closer detail apical lipid-raft compartments in enterocytes by immunogold electron microscopy and biochemical analyses. Novel membrane structures, deep-apical tubules, were visualized by the non-permeable surface marker Ruthenium Red in the brush-border region of the cells. The surface-connected tubules were labelled by antibodies to caveolin-1 and the glycolipid asialo G(M1), and they were sensitive to cholesterol depletion by methyl-beta-cyclodextrin, indicating the presence of raft microdomains. Deep-apical tubules were positioned close to the actin rootlets of adjacent microvilli in the terminal web region, which had a diameter of 50-100 nm, and penetrated up to 1 microm into the cytoplasm. Markers for transcytosis, IgA and the polymeric immunoglobulin receptor, as well as the resident brush-border enzyme aminopeptidase N, were present in these deep-apical tubules. We propose that deep-apical tubules are a specialized lipid-raft microdomain in the brush-border region functioning as a hub in membrane trafficking at the brush border. In addition, the sensitivity to cholesterol depletion suggests that deep-apical tubules function as a cell-surface membrane reservoir for cholesterol and for rapid adaptive changes in the size of microvilli at the brush border. PMID:12689332

  6. Targeting of Pseudorabies Virus Structural Proteins to Axons Requires Association of the Viral Us9 Protein with Lipid Rafts

    PubMed Central

    Lyman, Mathew G.; Curanovic, Dusica; Enquist, Lynn W.

    2008-01-01

    The pseudorabies virus (PRV) Us9 protein plays a central role in targeting viral capsids and glycoproteins to axons of dissociated sympathetic neurons. As a result, Us9 null mutants are defective in anterograde transmission of infection in vivo. However, it is unclear how Us9 promotes axonal sorting of so many viral proteins. It is known that the glycoproteins gB, gC, gD and gE are associated with lipid raft microdomains on the surface of infected swine kidney cells and monocytes, and are directed into the axon in a Us9-dependent manner. In this report, we determined that Us9 is associated with lipid rafts, and that this association is critical to Us9-mediated sorting of viral structural proteins. We used infected non-polarized and polarized PC12 cells, a rat pheochromocytoma cell line that acquires many of the characteristics of sympathetic neurons in the presence of nerve growth factor (NGF). In these cells, Us9 is highly enriched in detergent-resistant membranes (DRMs). Moreover, reducing the affinity of Us9 for lipid rafts inhibited anterograde transmission of infection from sympathetic neurons to epithelial cells in vitro. We conclude that association of Us9 with lipid rafts is key for efficient targeting of structural proteins to axons and, as a consequence, for directional spread of PRV from pre-synaptic to post-synaptic neurons and cells of the mammalian nervous system. PMID:18483549

  7. Amyloid-beta Alzheimer targets - protein processing, lipid rafts, and amyloid-beta pores.

    PubMed

    Arbor, Sage C; LaFontaine, Mike; Cumbay, Medhane

    2016-03-01

    Amyloid beta (Aβ), the hallmark of Alzheimer's Disease (AD), now appears to be deleterious in its low number aggregate form as opposed to the macroscopic Aβ fibers historically seen postmortem. While Alzheimer targets, such as the tau protein, amyloid precursor protein (APP) processing, and immune system activation continue to be investigated, the recent discovery that amyloid beta aggregates at lipid rafts and likely forms neurotoxic pores has led to a new paradigm regarding why past therapeutics may have failed and how to design the next round of compounds for clinical trials. An atomic resolution understanding of Aβ aggregates, which appear to exist in multiple conformations, is most desirable for future therapeutic development. The investigative difficulties, structures of these small Aβ aggregates, and current therapeutics are summarized in this review. PMID:27505013

  8. Amyloid-beta Alzheimer targets — protein processing, lipid rafts, and amyloid-beta pores

    PubMed Central

    Arbor, Sage C.; LaFontaine, Mike; Cumbay, Medhane

    2016-01-01

    Amyloid beta (Aβ), the hallmark of Alzheimer’s Disease (AD), now appears to be deleterious in its low number aggregate form as opposed to the macroscopic Aβ fibers historically seen postmortem. While Alzheimer targets, such as the tau protein, amyloid precursor protein (APP) processing, and immune system activation continue to be investigated, the recent discovery that amyloid beta aggregates at lipid rafts and likely forms neurotoxic pores has led to a new paradigm regarding why past therapeutics may have failed and how to design the next round of compounds for clinical trials. An atomic resolution understanding of Aβ aggregates, which appear to exist in multiple conformations, is most desirable for future therapeutic development. The investigative difficulties, structures of these small Aβ aggregates, and current therapeutics are summarized in this review. PMID:27505013

  9. Proteomic profiling of lipid rafts in a human breast cancer model of tumorigenic progression

    PubMed Central

    Caruso, Joseph A.; Stemmer, Paul M.

    2013-01-01

    Tumor biomarkers assist in the early detection of cancer, act as therapeutic targets for intervention, and function as diagnostic indicators for the evaluation of therapeutic responses. To identify novel human breast cancer biomarkers, we have analyzed the protein content of lipid rafts isolated from a series of human mammary epithelial cell lines with increasing tumorigenic potential. Since lipid rafts function as platforms for protein interaction critical to several biological processes, we hypothesized that the abundance of proteins associated with proliferation, invasion and metastasis would be dysregulated in highly transformed cells. For this purpose, the MCF10A epithelial lineage, which include benign MCF10A cells, premalignant AT and TG3B cells, and malignant CA1a tumor cells, was utilized. Detergent-resistant membranes were isolated from each line and proteins were identified and relatively quantitated using iTRAQ™ reagents and tandem mass spectrometry. 57 proteins were identified, and 1667 peptide identifications, mapping to 49 proteins, contained sufficient information for semi-quantitative analysis. When comparing malignant to benign cells, we observed consistent alterations in groups of proteins, such as a 5.7-fold average decrease in G protein content (n=5), 2.7-fold decrease glycosylphosphatidylinositol-linked proteins (n=7) and 3.3-fold increase in intermediate filaments (n=9). Several of the identified proteins, including caveolin-1, filamin A, keratins 5,6 & 17, and vimentin, are bona fide or candidate biomarkers in clinical studies, underscoring the usefulness of the MCF10A series as a model to better understand the biological mechanisms underlying cancer progression. PMID:21533873

  10. Heparin suppresses lipid raft-mediated signaling and ligand-independent EGF receptor activation.

    PubMed

    Liu, Yuan-Tao; Song, Lifang; Templeton, Douglas M

    2007-04-01

    Heparin is well known to suppress vascular smooth muscle cell (VSMC) proliferation, and attempts to exploit this therapeutically have led to recognition of multiple pathways for heparin's anti-mitogenic actions. At low concentrations (ca. 1 microg.ml(-1)), these suppressive effects may reflect physiological activities of endogenous heparan sulfates, and appear to be rapid responses to extracellular or cell surface-associated heparin. Because heparin has been shown to influence expression of caveolin proteins, and caveolae/lipid rafts are critical structures modulating cell signaling, we examined the effect of heparin on signaling involving cholesterol-rich membrane microdomains. The VSMC line PAC-1 activates the MAP kinase Erk in response to the cholesterol-sequestering agents methyl-beta-cyclodextrin and nystatin. This follows a temporal sequence that involves Ras-GTP activation of MEK, and is independent of PKC, Src, and PI3 kinase. However, ligand-independent phosphorylation of the EGF receptor (EGFR) by removal of cholesterol precedes Ras activation, and the EGFR kinase inhibitor AG1478 blocks Erk phosphorylation, supporting occurrence of the signaling sequence EGFR-Ras-MEK-Erk. Phosphorylation of EGFR occurs predominantly in caveolin-rich microdomains as identified by Western blotting of fractions from density gradient centrifugation of membranes prepared under detergent-free conditions. In these situations, heparin inhibits phosphorylation of EGFR on the Src-dependent site Tyr(845), but not the autophosphorylation of Tyr(1173), and decreases Ras activation and Erk phosphorylation. We conclude that heparin can suppress Erk signaling in VSMC with effects on site-specific phosphorylation of EGFR localized in caveolin-enriched lipid rafts. PMID:17226785

  11. Lipid rafts association and anti-apoptotic function of prohibitin in ultraviolet B light-irradiated HaCaT keratinocytes.

    PubMed

    Wu, Qiong; Wu, Shiyong

    2012-08-01

    Upon UVB irradiation, an alternation of major lipid raft components can lead to the recruitment/activation of rafts-associated proteins and initiation of downstream apoptotic signalling pathways. We used two-dimensional gel electrophoresis (2-DE) to identify potential regulators of UVB-induced apoptosis and mass spectrometry fingerprint analysis to identify proteins that are altered in the rafts after UVB irradiation. Our data show that levels of several proteins, including prohibitin (PHB), were changed in lipid rafts after UVB irradiation. We also demonstrate that while total PHB expression was not changed, the protein was enriched in lipid rafts after UVB irradiation. Reduced expression of PHB using siRNA knockdown resulted in an increase in cellular apoptosis after UVB irradiation. Based on these results, we propose that PHB protects keratinocytes from UVB-induced apoptosis. PMID:22776003

  12. 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

  13. Sustained Epigenetic Drug Delivery Depletes Cholesterol-Sphingomyelin Rafts from Resistant Breast Cancer Cells, Influencing Biophysical Characteristics of Membrane Lipids.

    PubMed

    Raghavan, Vijay; Vijayaraghavalu, Sivakumar; Peetla, Chiranjeevi; Yamada, Masayoshi; Morisada, Megan; Labhasetwar, Vinod

    2015-10-27

    Cell-membrane lipid composition can greatly influence biophysical properties of cell membranes, affecting various cellular functions. We previously showed that lipid synthesis becomes altered in the membranes of resistant breast cancer cells (MCF-7/ADR); they form a more rigid, hydrophobic lipid monolayer than do sensitive cell membranes (MCF-7). These changes in membrane lipids of resistant cells, attributed to epigenetic aberration, significantly affected drug transport and endocytic function, thus impacting the efficacy of anticancer drugs. The present study's objective was to determine the effects of the epigenetic drug, 5-aza-2'-deoxycytidine (DAC), delivered in sustained-release nanogels (DAC-NGs), on the composition and biophysical properties of membrane lipids of resistant cells. Resistant and sensitive cells were treated with DAC in solution (DAC-sol) or DAC-NGs, and cell-membrane lipids were isolated and analyzed for lipid composition and biophysical properties. In resistant cells, we found increased formation of cholesterol-sphingomyelin (CHOL-SM) rafts with culturing time, whereas DAC treatment reduced their formation. In general, the effect of DAC-NGs was greater in changing the lipid composition than with DAC-sol. DAC treatment also caused a rise in levels of certain phospholipids and neutral lipids known to increase membrane fluidity, while reducing the levels of certain lipids known to increase membrane rigidity. Isotherm data showed increased lipid membrane fluidity following DAC treatment, attributed to decrease levels of CHOL-SM rafts (lamellar beta [Lβ] structures or ordered gel) and a corresponding increase in lipids that form lamellar alpha-structures (Lα, liquid crystalline phase). Sensitive cells showed marginal or insignificant changes in lipid profile following DAC-treatment, suggesting that epigenetic changes affecting lipid biosynthesis are more specific to resistant cells. Since membrane fluidity plays a major role in drug transport

  14. 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

  15. Induction of apoptosis by the ginsenoside Rh2 by internalization of lipid rafts and caveolae and inactivation of Akt

    PubMed Central

    Park, E-K; Lee, EJ; Lee, S-H; Koo, KH; Sung, JY; Hwang, EH; Park, JH; Kim, C-W; Jeong, K-C; Park, B-K; Kim, Y-N

    2010-01-01

    Background and purpose: Lipid rafts and caveolae are membrane microdomains with important roles in cell survival signalling involving the Akt pathway. Cholesterol is important for the structure and function of these microdomains. The ginsenoside Rh2 exhibits anti-tumour activity. Because Rh2 is structurally similar to cholesterol, we investigated the possibility that Rh2 exerted its anti-tumour effect by modulating rafts and caveolae. Experimental approach: A431 cells (human epidermoid carcinoma cell line) were treated with Rh2 and the effects on cell apoptosis, raft localization and Akt activation measured. We also examined the effects of over-expression of Akt and active-Akt on Rh2-induced cell death. Key results: Rh2 induced apoptosis concentration- and time-dependently. Rh2 reduced the levels of rafts and caveolae in the plasma membrane and increased their internalization. Furthermore, Akt activity was decreased and consequently, Akt-dependent phosphorylation of Bad, a pro-survival protein, was decreased whereas the pro-apoptotic proteins, Bim and Bax, were increased upon Rh2 treatment. Unlike microdomain internalization induce by cholesterol depletion, Rh2-mediated internalization of rafts and caveolae was not reversed by cholesterol addition. Also, cholesterol addition did not restore Akt activation or rescue cells from Rh2-induced cell death. Rh2-induced cell death was attenuated in MDA-MB-231 cells over-expressing either wild-type or dominant-active Akt. Conclusions and implications: Rh2 induced internalization of rafts and caveolae, leading to Akt inactivation, and ultimately apoptosis. Because elevated levels of membrane rafts and caveolae, and Akt activation have been correlated with cancer development, internalization of these microdomains by Rh2 could potentially be used as an anti-cancer therapy. PMID:20590613

  16. Ceramide inhibits PKCθ by regulating its phosphorylation and translocation to lipid rafts in Jurkat cells.

    PubMed

    Hage-Sleiman, Rouba; Hamze, Asmaa B; El-Hed, Aimée F; Attieh, Randa; Kozhaya, Lina; Kabbani, Sarah; Dbaibo, Ghassan

    2016-08-01

    Protein kinase C theta (PKCθ) is a novel, calcium-independent member of the PKC family of kinases that was identified as a central player in T cell signaling and proliferation. Upon T cell activation by antigen-presenting cells, PKCθ gets phosphorylated and activated prior to its translocation to the immunological synapse where it couples with downstream effectors. PKCθ may be regulated by ceramide, a crucial sphingolipid that is known to promote differentiation, growth arrest, and apoptosis. To further investigate the mechanism, we stimulated human Jurkat T cells with either PMA or anti-CD3/anti-CD28 antibodies following induction of ceramide accumulation by adding exogenous ceramide, bacterial sphingomyelinase, or Fas ligation. Our results suggest that ceramide regulates the PKCθ pathway through preventing its critical threonine 538 (Thr538) phosphorylation and subsequent activation, thereby inhibiting the kinase's translocation to lipid rafts. Moreover, this inhibition is not likely to be a generic effect of ceramide on membrane reorganization. Other lipids, namely dihydroceramide, palmitate, and sphingosine, did not produce similar effects on PKCθ. Addition of the phosphatase inhibitors okadaic acid and calyculin A reversed the inhibition exerted by ceramide, and this suggests involvement of a ceramide-activated protein phosphatase. Such previously undescribed mechanism of regulation of PKCθ raises the possibility that ceramide, or one of its derivatives, and may prove valuable in novel therapeutic approaches for disorders involving autoimmunity or excessive inflammation-where PKCθ plays a critical role. PMID:26798039

  17. Bilayer Asymmetry Influences Integrin Sequestering in Raft-Mimicking Lipid Mixtures

    PubMed Central

    Hussain, Noor F.; Siegel, Amanda P.; Ge, Yifan; Jordan, Rainer; Naumann, Christoph A.

    2013-01-01

    There is growing recognition that lipid heterogeneities in cellular membranes play an important role in the distribution and functionality of membrane proteins. However, the detection and characterization of such heterogeneities at the cellular level remains challenging. Here we report on the poorly understood relationship between lipid bilayer asymmetry and membrane protein sequestering in raft-mimicking model membrane mixtures using a powerful experimental platform comprised of confocal spectroscopy XY-scan and photon-counting histogram analyses. This experimental approach is utilized to probe the domain-specific sequestering and oligomerization state of αvβ3 and α5β1 integrins in bilayers, which contain coexisting liquid-disordered/liquid-ordered (ld/lo) phase regions exclusively in the top leaflet of the bilayer (bottom leaflet contains ld phase). Comparison with previously reported integrin sequestering data in bilayer-spanning lo-ld phase separations demonstrates that bilayer asymmetry has a profound influence on αvβ3 and α5β1 sequestering behavior. For example, both integrins sequester preferentially to the lo phase in asymmetric bilayers, but to the ld phase in their symmetric counterparts. Furthermore, our data show that bilayer asymmetry significantly influences the role of native ligands in integrin sequestering. PMID:23708361

  18. Structure of Cholesterol/Ceramide Monolayer Mixtures: Implications to the Molecular Organization of Lipid Rafts

    PubMed Central

    Scheffer, Luana; Solomonov, Inna; Weygand, Markus Jan; Kjaer, Kristian; Leiserowitz, Leslie; Addadi, Lia

    2005-01-01

    The structure of monolayers of cholesterol/ceramide mixtures was investigated using grazing incidence x-ray diffraction, immunofluorescence, and atomic force microscopy techniques. Grazing incidence x-ray diffraction measurements showed the existence of a crystalline mixed phase of the two components within a range of compositions of cholesterol/ceramide between 100:0 and 67:33. The mixed phase coexists with the ceramide crystalline phase in the range of compositions between 50:50 and 30:70; between 30:70 and 0:100 only the highly crystalline phase of ceramide was detected. The latter was determined and modeled. Immunolabeling was performed with an antibody specific to the cholesterol monohydrate crystalline arrangement. The antibody recognizes crystalline cholesterol monolayers, but does not interact with crystalline ceramide. Immunofluorescence and atomic force microscopy data show that in uncompressed ceramide monolayers, the highly crystalline phase coexists with a disordered loosely packed phase. In contrast, no disordered phase coexists with the new crystalline mixed phase. We conclude that the new mixed phase represents a stable homogeneous arrangement of cholesterol with ceramide. As ceramide incorporates the lipid backbone common to all sphingolipids, this arrangement may be relevant to the understanding of the molecular organization of lipid rafts. PMID:15722431

  19. Nucleolin-mediated cellular trafficking of DNA nanoparticle is lipid raft and microtubule dependent and can be modulated by glucocorticoid.

    PubMed

    Chen, Xuguang; Shank, Samuel; Davis, Pamela B; Ziady, Assem G

    2011-01-01

    DNA nanoparticles (DNPs) are nonviral gene transfer vectors with excellent in vivo potential. Previously, we reported that cell surface nucleolin directly binds DNPs, and functions as an important receptor for DNPs. However, the fate of the nucleolin-DNP complex following cellular uptake remains elusive. In this study, we examined the role of lipid rafts in the uptake of DNPs, and found that both nucleolin and DNPs are recovered from the low-density raft fractions of the sucrose gradient. Furthermore, nucleolin colocalizes with, and coimmunoprecipitates with a raft protein, flotillin. Disruption of lipid rafts by depleting membrane cholesterol significantly inhibited DNP transfection, while inhibition of other endocytic pathways had little effect. Following the uptake, the nuclear import of the DNPs required microtubules but not F-actin. By coimmunoprecipitation in conjunction with tandem mass spectrometry, we identified glucocorticoid receptor (GCR) as a nucleolin-associated protein, and confirmed this result by western blot. Cortisone or dexamethasone increased nucleolin's association with GCR, and transfection by DNPs. Finally, we detected the expression of nucleolin on the surface of airway epithelia in vivo. Taken together, our findings shed light on important determinants of DNP trafficking in cells and support the notion that nucleolin is a good target for nonviral gene delivery. PMID:20959809

  20. Investigating lipid interactions and the process of raft formation in cellular membranes using ToF-SIMS

    NASA Astrophysics Data System (ADS)

    McQuaw, Carolyn M.; Sostarecz, Audra G.; Zheng, Leiliang; Ewing, Andrew G.; Winograd, Nicholas

    2006-07-01

    There is an increased interest in how lipids interact with each other, especially in the lateral separation of lipids into coexisting liquid phases as this is believed to be an attribute of raft formation in cell membranes. ToF-SIMS has shown itself to be an excellent tool for investigating cellular and model membrane systems and will be perhaps the most powerful one for investigating raft formation. Results from our laboratory show the capability of ToF-SIMS at identifying unequivocally the content of coexisting liquid lipid phases. Using supported lipid monolayers we find that the inclusion of dipalmitoylphosphatidylethanolamine (DPPE) to a homogeneous dipalmitoyl-phosphatidylcholine (DPPC)/cholesterol phase results in the formation of cholesterol-rich domains [A.G. Sostarecz, C.M. McQuaw, A.G. Ewing, N. Winograd, J. Am. Chem. Soc. 126 (2004) 13882]. Also, for DPPE/cholesterol systems a single homogeneous DPPE/cholesterol phase is formed at ˜50 mol% cholesterol, whereas DPPC/cholesterol systems form a single phase at 30 mol% cholesterol [C.M. McQuaw, A. Sostarecz, L. Zheng, A.G. Ewing, N. Winograd, Langmuir 21 (2005) 807]. Currently we are exploring the incorporation of sphingomyelin into phospholipid-cholesterol mixtures in an effort to gain a better understanding of its role in raft formation.

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

    SciTech Connect

    Eum, Sung Yong Jaraki, Dima; András, Ibolya E.; Toborek, Michal

    2015-09-15

    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) 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-associated PP

  2. Surfactant lipids regulate LPS-induced interleukin-8 production in A549 lung epithelial cells by inhibiting translocation of TLR4 into lipid raft domains

    PubMed Central

    Abate, Wondwossen; Alghaithy, Abdulaziz A.; Parton, Joan; Jones, Kenneth P.; Jackson, Simon K.

    2010-01-01

    In addition to providing mechanical stability, growing evidence suggests that surfactant lipid components can modulate inflammatory responses in the lung. However, little is known of the molecular mechanisms involved in the immunomodulatory action of surfactant lipids. This study investigates the effect of the lipid-rich surfactant preparations Survanta®, Curosurf®, and the major surfactant phospholipid dipalmitoylphosphatidylcholine (DPPC) on interleukin-8 (IL-8) gene and protein expression in human A549 lung epithelial cells using immunoassay and PCR techniques. To examine potential mechanisms of the surfactant lipid effects, Toll-like receptor 4 (TLR4) expression was analyzed by flow cytometry, and membrane lipid raft domains were separated by density gradient ultracentrifugation and analyzed by immunoblotting with anti-TLR4 antibody. The lipid-rich surfactant preparations Survanta®, Curosurf®, and DPPC, at physiological concentrations, significantly downregulated lipopolysaccharide (LPS)-induced IL-8 expression in A549 cells both at the mRNA and protein levels. The surfactant preparations did not affect the cell surface expression of TLR4 or the binding of LPS to the cells. However, LPS treatment induced translocation of TLR4 into membrane lipid raft microdomains, and this translocation was inhibited by incubation of the cells with the surfactant lipid. This study provides important mechanistic details of the immune-modulating action of pulmonary surfactant lipids. PMID:19648651

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

    PubMed

    Eum, Sung Yong; Jaraki, Dima; András, Ibolya E; Toborek, Michal

    2015-09-15

    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 1h. Moreover, lipid rafts modulated the reduction of occludin level through activation of matrix metalloproteinase 2 (MMP-2) after 24h 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. PMID:26080028

  4. Cholesterol lipids of Borrelia burgdorferi form lipid rafts and are required for the bactericidal mechanism of a complement-independent antibody

    PubMed Central

    LaRocca, Timothy J; Crowley, Jameson T; Cusack, Brian J; Pathak, Priyadarshini; Benach, Jordi; London, Erwin; Garcia-Monco, Juan C; Benach, Jorge L

    2010-01-01

    SUMMARY Borrelia burgdorferi (the agent of Lyme disease) is unusual in that it contains free cholesterol and cholesterol glycolipids. It is also susceptible to complement-independent bactericidal antibodies, such as CB2, a monoclonal IgG1 against outer surface protein B (OspB). The bactericidal action of CB2 requires the presence of cholesterol glycolipids and cholesterol. Through ultrastructural, biochemical and biophysical approaches, we show that these cholesterol glycolipids exist as lipid raft-like microdomains in the outer membrane of cultured and mouse-derived B. burgdorferi, and in model membranes from B. burgdorferi lipids. The order and size of the microdomains of intact cells and model membranes are temperature sensitive and correlate with the bactericidal activity of CB2. Here we demonstrate the existence of cholesterol-containing lipid raft-like microdomains in a prokaryote. PMID:20951967

  5. 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. PMID:27422101

  6. Non-genomic inhibitory effect of glucocorticoids on activated peripheral blood basophils through suppression of lipid raft formation.

    PubMed

    Yamagata, S; Tomita, K; Sano, H; Itoh, Y; Fukai, Y; Okimoto, N; Watatani, N; Inbe, S; Miyajima, H; Tsukamoto, K; Santoh, H; Ichihashi, H; Sano, A; Sato, R; Tohda, Y

    2012-10-01

    We investigated the non-genomic effects of glucocorticoids (GCs) on inhibition of plasma membrane lipid raft formation in activated human basophils. Human basophils obtained from house dust mite (HDM)-sensitive volunteers were pretreated with hydrocortisone (CORT) or dexamethasone (Dex) for 30 min and then primed with phorbol 12-myristate 13-acetate (PMA, 10 ng/ml) or HDM (10 µg/ml). The expression of CD63, a basophil activation marker, was assessed by flow cytometry. Membrane-bound GC receptors (mGCRs) were analysed by flow cytometry and confocal laser microscopy. Lipid rafts were assessed using a GM1 ganglioside probe and visualization by confocal laser microscopy. Pretreatment of basophils with CORT (10(-4) M and 10(-5) M) and Dex (10(-7) M) significantly inhibited CD63 expression 20 min after addition of PMA or HDM. The inhibitory effects of GCs were not altered by the nuclear GC receptor (GCR) antagonist RU486 (10(-5) M) or the protein synthesis inhibitor cycloheximide (10(-4) M) (P < 0·05). CORT coupled to bovine serum albumin (BSA-CORT) mimicked the rapid inhibitory effects of CORT, suggesting the involvement of mGCRs. mGCRs were detectable on the plasma membrane of resting basophils and formed nanoclusters following treatment with PMA or HDM. Pretreatment of cells with BSA-CORT inhibited the expression of mGCRs and nanoclustering of ganglioside GM1 in lipid rafts. The study provides evidence that non-genomic mechanisms are involved in the rapid inhibitory effect of GCs on the formation of lipid raft nanoclusters, through binding to mGCRs on the plasma membrane of activated basophils. PMID:22943204

  7. 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

  8. Caspase-8 and c-FLIPL Associate in Lipid Rafts with NF-κB Adaptors during T Cell Activation*

    PubMed Central

    Misra, Ravi S.; Russell, Jennifer Q.; Koenig, Andreas; Hinshaw-Makepeace, Jennifer A.; Wen, Renren; Wang, Demin; Huo, Hairong; Littman, Dan R.; Ferch, Uta; Ruland, Jurgen; Thome, Margot; Budd, Ralph C.

    2015-01-01

    Humans and mice lacking functional caspase-8 in T cells manifest a profound immunodeficiency syndrome due to defective T cell antigen receptor (TCR)-induced NF-κB signaling and proliferation. It is unknown how caspase-8 is activated following T cell stimulation, and what is the caspase-8 substrate(s) that is necessary to initiate T cell cycling. We observe that following TCR ligation, a small portion of total cellular caspase-8 and c-FLIPL rapidly migrate to lipid rafts where they associate in an active caspase complex. Activation of caspase-8 in lipid rafts is followed by rapid cleavage of c-FLIPL at a known caspase-8 cleavage site. The active caspase·c-FLIP complex forms in the absence of Fas (CD95/APO1) and associates with the NF-κB signaling molecules RIP1, TRAF2, and TRAF6, as well as upstream NF-κB regulators PKCθ, CARMA1, Bcl-10, and MALT1, which connect to the TCR. The lack of caspase-8 results in the absence of MALT1 and Bcl-10 in the active caspase complex. Consistent with this observation, inhibition of caspase activity attenuates NF-κB activation. The current findings define a link among TCR, caspases, and the NF-κB pathway that occurs in a sequestered lipid raft environment in T cells. PMID:17462996

  9. Effect of Receptor Dimerization on Membrane Lipid Raft Structure Continuously Quantified on Single Cells by Camera Based Fluorescence Correlation Spectroscopy

    PubMed Central

    Jin, Weixiang; Pralle, Arnd

    2015-01-01

    Membrane bound cell signaling is modulated by the membrane ultra-structure, which itself may be affected by signaling. However, measuring the interaction of membrane proteins with membrane structures in intact cells in real-time poses considerable challenges. In this paper we present a non-destructive fluorescence method that quantifies these interactions in single cells, and is able to monitor the same cell continuously to observe small changes. This approach combines total internal fluorescence microscopy with fluorescence correlation spectroscopy to measure the protein’s diffusion and molecular concentration in different sized areas simultaneously. It correctly differentiates proteins interacting with membrane fences from proteins interacting with cholesterol-stabilized domains, or lipid rafts. This method detects small perturbations of the membrane ultra-structure or of a protein’s tendency to dimerize. Through continuous monitoring of single cells, we demonstrate how dimerization of GPI-anchored proteins increases their association with the structural domains. Using a dual-color approach we study the effect of dimerization of one GPI-anchored protein on another type of GPI-anchored protein expressed in the same cell. Scans over the cell surface reveal a correlation between cholesterol stabilized domains and membrane cytoskeleton. PMID:25811483

  10. Effects of Leucin-Enkephalins on Surface Characteristics and Morphology of Model Membranes Composed of Raft-Forming Lipids.

    PubMed

    Tsanova, Asya; Jordanova, A; Lalchev, Z

    2016-06-01

    During the last decades opioid peptides, like enkephalins (Tyr-Gly-Gly-Phe-Met/Leu) are subject to extensive studies due to their antinociceptive action in organism. According to the membrane catalysis theory, in order to adopt a proper conformation for binding to their receptors, opioid peptides interact with the lipid phase of the membrane receptor surrounding. With this regard, the aim of the present work was to study the effects of synthetic leucine-enkephalin and leucine-enkephalinamide on surface characteristics and morphology of lipid monolayers, composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, sphingomyelin, and cholesterol alone and with their mixtures. The lipids were chosen to represent a model of a membrane raft, since it is known that G-protein-coupled receptors, including opioid receptors, are located preferably in membrane rafts. By using Langmuir's monolayer method, the change in surface pressure of the model membranes before and after the addition of the synthetic enkephalins was studied, and the compressional moduli of the lipids and lipid-peptides monolayers were determined. In addition, by Brewster angle microscopy, the surface morphology of the lipid monolayers alone and after the injection of both enkephalins was monitored. Our results showed that both leucine-enkephalins affected the lipid monolayers surface characteristics, and led to an increase in surface density of the mixed surface lipids/enkephalins films at loose lipid packing. This effect was more pronounced for the enkephalinamide, suggesting a different mechanism of interaction for the amidated enkephalin with the lipid phase, as compared to leucine-enkephalin. PMID:26661722

  11. REEP2 Enhances Sweet Receptor Function by Recruitment to Lipid Rafts

    PubMed Central

    Ilegems, Erwin; Iwatsuki, Ken; Kokrashvili, Zaza; Benard, Outhiriaradjou; Ninomiya, Yuzo; Margolskee, Robert F.

    2010-01-01

    Heterologously expressed sensory receptors generally do not achieve the ligand sensitivity observed in vivo, and may require specific accessory proteins to ensure optimal function. We searched for taste cell-expressed receptor transporting protein (RTP) and receptor expression enhancing protein (REEP) family members that might serve as accessory molecules to enhance gustatory receptor function. We determined that REEP2 is an integral membrane protein expressed in taste cells, physically associates with both subunits of the type 1 taste receptor 2 and type 1 taste receptor 3 sweet receptor and specifically enhances responses to tastants of heterologously expressed sweet and bitter taste receptors. Downregulation of endogenously expressed REEP2 in the chemosensory enteroendocrine GLUTag cell line dramatically reduced sensitivity of endogenous sweet receptors. In contrast to the observation that RTP1, RTP2, and REEP1 enhance function of olfactory receptors by promoting their transit to the cell surface, we found that REEP2 does not increase cell surface expression of sweet receptors but instead alters their spatial organization. REEP2 recruits sweet receptors into lipid raft microdomains localized near the taste cell’s apical region, thereby improving G-protein-coupled receptor signaling and promoting receptor access to tastants arriving through the apical taste pore. PMID:20943918

  12. Segregation of leading-edge and uropod components into specific lipid rafts during T cell polarization

    PubMed Central

    Gómez-Moutón, Concepción; Abad, Jose Luis; Mira, Emilia; Lacalle, Rosa Ana; Gallardo, Eduard; Jiménez-Baranda, Sonia; Illa, Isabel; Bernad, Antonio; Mañes, Santos; Martínez-A., Carlos

    2001-01-01

    Redistribution of specialized molecules in migrating cells develops asymmetry between two opposite cell poles, the leading edge and the uropod. We show that acquisition of a motile phenotype in T lymphocytes results in the asymmetric redistribution of ganglioside GM3- and GM1-enriched raft domains to the leading edge and to the uropod, respectively. This segregation to each cell pole parallels the specific redistribution of membrane proteins associated to each raft subfraction. Our data suggest that raft partitioning is a major determinant for protein redistribution in polarized T cells, as ectopic expression of raft-associated proteins results in their asymmetric redistribution, whereas non-raft-partitioned mutants of these proteins are distributed homogeneously in the polarized cell membrane. Both acquisition of a migratory phenotype and SDF-1α-induced chemotaxis are cholesterol depletion-sensitive. Finally, GM3 and GM1 raft redistribution requires an intact actin cytoskeleton, but is insensitive to microtubule disruption. We propose that membrane protein segregation not only between raft and nonraft domains but also between distinct raft subdomains may be an organizational principle that mediates redistribution of specialized molecules needed for T cell migration. PMID:11493690

  13. 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

  14. The Deleterious Effects of Oxidative and Nitrosative Stress on Palmitoylation, Membrane Lipid Rafts and Lipid-Based Cellular Signalling: New Drug Targets in Neuroimmune Disorders.

    PubMed

    Morris, Gerwyn; Walder, Ken; Puri, Basant K; Berk, Michael; Maes, Michael

    2016-09-01

    Oxidative and nitrosative stress (O&NS) is causatively implicated in the pathogenesis of Alzheimer's and Parkinson's disease, multiple sclerosis, chronic fatigue syndrome, schizophrenia and depression. Many of the consequences stemming from O&NS, including damage to proteins, lipids and DNA, are well known, whereas the effects of O&NS on lipoprotein-based cellular signalling involving palmitoylation and plasma membrane lipid rafts are less well documented. The aim of this narrative review is to discuss the mechanisms involved in lipid-based signalling, including palmitoylation, membrane/lipid raft (MLR) and n-3 polyunsaturated fatty acid (PUFA) functions, the effects of O&NS processes on these processes and their role in the abovementioned diseases. S-palmitoylation is a post-translational modification, which regulates protein trafficking and association with the plasma membrane, protein subcellular location and functions. Palmitoylation and MRLs play a key role in neuronal functions, including glutamatergic neurotransmission, and immune-inflammatory responses. Palmitoylation, MLRs and n-3 PUFAs are vulnerable to the corruptive effects of O&NS. Chronic O&NS inhibits palmitoylation and causes profound changes in lipid membrane composition, e.g. n-3 PUFA depletion, increased membrane permeability and reduced fluidity, which together lead to disorders in intracellular signal transduction, receptor dysfunction and increased neurotoxicity. Disruption of lipid-based signalling is a source of the neuroimmune disorders involved in the pathophysiology of the abovementioned diseases. n-3 PUFA supplementation is a rational therapeutic approach targeting disruptions in lipid-based signalling. PMID:26310971

  15. Membrane lipid rafts and neurobiology: age-related changes in membrane lipids and loss of neuronal function.

    PubMed

    Egawa, Junji; Pearn, Matthew L; Lemkuil, Brian P; Patel, Piyush M; Head, Brian P

    2016-08-15

    A better understanding of the cellular physiological role that plasma membrane lipids, fatty acids and sterols play in various cellular systems may yield more insight into how cellular and whole organ function is altered during the ageing process. Membrane lipid rafts (MLRs) within the plasma membrane of most cells serve as key organizers of intracellular signalling and tethering points of cytoskeletal components. MLRs are plasmalemmal microdomains enriched in sphingolipids, cholesterol and scaffolding proteins; they serve as a platform for signal transduction, cytoskeletal organization and vesicular trafficking. Within MLRs are the scaffolding and cholesterol binding proteins named caveolin (Cav). Cavs not only organize a multitude of receptors including neurotransmitter receptors (NMDA and AMPA receptors), signalling proteins that regulate the production of cAMP (G protein-coupled receptors, adenylyl cyclases, phosphodiesterases (PDEs)), and receptor tyrosine kinases involved in growth (Trk), but also interact with components that modulate actin and tubulin cytoskeletal dynamics (e.g. RhoGTPases and actin binding proteins). MLRs are essential for the regulation of the physiology of organs such as the brain, and age-related loss of cholesterol from the plasma membrane leads to loss of MLRs, decreased presynaptic vesicle fusion, and changes in neurotransmitter release, all of which contribute to different forms of neurodegeneration. Thus, MLRs provide an active membrane domain that tethers and reorganizes the cytoskeletal machinery necessary for membrane and cellular repair, and genetic interventions that restore MLRs to normal cellular levels may be exploited as potential therapeutic means to reverse the ageing and neurodegenerative processes. PMID:26332795

  16. L-plastin is involved in NKG2D recruitment into lipid rafts and NKG2D-mediated NK cell migration.

    PubMed

    Serrano-Pertierra, Esther; Cernuda-Morollón, Eva; Brdička, Tomáš; Hoøejši, Václav; López-Larrea, Carlos

    2014-09-01

    Membrane rafts are microdomains of the plasma membrane that have multiple biological functions. The involvement of these structures in the biology of T cells, namely in signal transduction by the TCR, has been widely studied. However, the role of membrane rafts in immunoreceptor signaling in NK cells is less well known. We studied the distribution of the activating NKG2D receptor in lipid rafts by isolating DRMs in a sucrose density gradient or by raft fractionation by β-OG-selective solubility in the NKL cell line. We found that the NKG2D-DAP10 complex and pVav are recruited into rafts upon receptor stimulation. Qualitative proteomic analysis of these fractions showed that the actin cytoskeleton is involved in this process. In particular, we found that the actin-bundling protein L-plastin plays an important role in the clustering of NKG2D into lipid rafts. Moreover, coengagement of the inhibitory receptor NKG2A partially disrupted NKG2D recruitment into rafts. Furthermore, we demonstrated that L-plastin participates in NKG2D-mediated inhibition of NK cell chemotaxis. PMID:24803550

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

    SciTech Connect

    Valuckaite, V.; Zaborina, O.; Long, J.; Hauer-Jensen, M.; Wang, J.; Holbrook, C.; Zaborin, A.; Drabik, K.; Katdare, M.; Mauceri, H.; Weichselbaum, R.; Firestone, M. A.; Lee, K. Y.; Chang, E. B.; Matthews, J.; Alverdy, J. C.; Materials Science Division; Univ. of Chicago; Univ. of Arkansas

    2009-12-01

    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 surgically 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.

  18. Clustering of Neuronal K+-Cl− Cotransporters in Lipid Rafts by Tyrosine Phosphorylation*

    PubMed Central

    Watanabe, Miho; Wake, Hiroaki; Moorhouse, Andrew J.; Nabekura, Junichi

    2009-01-01

    The neuronal K+-Cl− cotransporter (KCC2) is a membrane transport protein that extrudes Cl− from neurons and helps maintain low intracellular [Cl−] and hyperpolarizing GABAergic synaptic potentials. Depolarizing γ-aminobutyric acid (GABA) responses in neonatal neurons and following various forms of neuronal injury are associated with reduced levels of KCC2 expression. Despite the importance for plasticity of inhibitory transmission, less is known about cellular mechanisms involved in more dynamic changes in KCC2 function. In this study, we investigated the role of tyrosine phosphorylation in KCC2 localization and function in hippocampal neurons and in cultured GT1-7 cells. Mutation to the putative tyrosine phosphorylation site within the long intracellular carboxyl terminus of KCC2(Y1087D) or application of the tyrosine kinase inhibitor genistein shifted the GABA reversal potential (EGABA) to more depolarized values, indicating reduced KCC2 function. This was associated with a change in the expression pattern of KCC2 from a punctate distribution to a more uniform distribution, suggesting that functional tyrosine-phosphorylated KCC2 forms clusters in restricted membrane domains. Sodium vanadate, a tyrosine phosphatase inhibitor, increased the proportion of KCC2 associated with lipid rafts membrane domains. Loss of tyrosine phosphorylation also reduced oligomerization of KCC2. A loss of the punctuate distribution and oligomerization of KCC2 and a more depolarized EGABA were seen when the 28-amino-acid carboxyl terminus of KCC2 was deleted. These results indicate that direct tyrosine phosphorylation of KCC2 results in membrane clusters and functional transport activity, suggesting a mechanism by which intracellular Cl− concentrations and GABA responses can be rapidly modulated. PMID:19679663

  19. Visfatin-Induced Lipid Raft Redox Signaling Platforms and Dysfunction in Glomerular Endothelial Cells

    PubMed Central

    Boini, Krishna M.; Zhang, Chun; Xia, Min; Han, Wei-Qing; Brimson, Christopher; Poklis, Justin L.; Li, Pin-Lan

    2010-01-01

    Adipokines have been reported to contribute to glomerular injury during obesity or diabetes mellitus. However, the mechanisms mediating the actions of various adipokines on the kidney remained elusive. The present study was performed to determine whether acid sphingomyelinase (ASM)-ceramide associated lipid raft (LR) clustering is involved in local oxidative stress in glomerular endothelial cells (GECs) induced by adipokines such as visfatin and adiponectin. Using confocal microscopy, visfatin but not adiponectin was found to increase LRs clustering in the membrane of GECs in a dose and time dependent manner. Upon visfatin stimulation ASMase activity was increased, and an aggregation of ASMase product, ceramide and NADPH oxidase subunits, gp91phox and p47phox were observed in the LR clusters, forming a LR redox signaling platform. The formation of this signaling platform was blocked by prior treatment with LR disruptor filipin, ASMase inhibitor amitriptyline, ASMase siRNA, gp91phox siRNA and adiponectin. Corresponding to LR clustering and aggregation of NADPH subunits, superoxide (O2•−) production was significantly increased (2.7 folds) upon visfatin stimulation, as measured by electron spin resonance (ESR) spectrometry. Functionally, visfatin significantly increased the permeability of GEC layer in culture and disrupted microtubular networks, which were blocked by inhibition of LR redox signaling platform formation. In conclusion, the injurious effect of visfatin, but not adiponectin on the glomerular endothelium is associated with the formation of LR redox signaling platforms via LR clustering, which produces local oxidative stress resulting in the disruption of microtubular networks in GECs and increases the glomerular permeability. PMID:20858552

  20. The novel chlamydial adhesin CPn0473 mediates the lipid raft-dependent uptake of Chlamydia pneumoniae.

    PubMed

    Fechtner, Tim; Galle, Jan N; Hegemann, Johannes H

    2016-08-01

    Chlamydiae are Gram-negative, obligate intracellular pathogens that pose a serious threat to public health worldwide. Chlamydial surface molecules are essential for host cell invasion. The first interaction with the host cell is thereby accomplished by the Outer membrane complex protein B (OmcB) binding to heparan sulfate moieties on the host cell surface, followed by the interaction of the chlamydial polymorphic membrane proteins (Pmps) with host cell receptors. Specifically, the interaction of the Pmp21 adhesin and invasin with its human interaction partner, the epidermal growth factor receptor, results in receptor activation, down-stream signalling and finally internalization of the bacteria. Blocking both, the OmcB and Pmp21 adhesion pathways, did not completely abolish infection, suggesting the presence of additional factors relevant for host cell invasion. Here, we show that the novel surface protein CPn0473 of Chlamydia pneumoniae contributes to the binding and invasion of infectious chlamydial particles. CPn0473 is expressed late in the infection cycle and located on the infectious chlamydial cell surface. Soluble recombinant CPn0473 as well as rCPn0473-coupled fluorescent latex beads adhere to human epithelial HEp-2 cells. Interestingly, in classical infection blocking experiments pretreatment of HEp-2 cells with rCPn0473 does not attenuate adhesion but promotes dose-dependently internalization by C. pneumoniae suggesting an unusual mode of action for this adhesin. This CPn0473-dependent promotion of infection by C. pneumoniae depends on two different domains within the protein and requires intact lipid rafts. Thus, inhibition of the interaction of CPn0473 with the host cell could provide a way to reduce the virulence of C. pneumoniae. PMID:26780295

  1. 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

  2. Resistance to alkyl-lysophospholipid-induced apoptosis due to downregulated sphingomyelin synthase 1 expression with consequent sphingomyelin- and cholesterol-deficiency in lipid rafts

    PubMed Central

    Van der Luit, Arnold H.; Budde, Marianne; Zerp, Shuraila; Caan, Wendy; Klarenbeek, Jeffrey B.; Verheij, Marcel; van Blitterswijk, Wim J.

    2006-01-01

    The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; Et-18-OCH3) induces apoptosis in S49 mouse lymphoma cells. To this end, ALP is internalized by lipid raft-dependent endocytosis and inhibits phosphatidylcholine synthesis. A variant cell-line, S49AR, which is resistant to ALP, was shown previously to be unable to internalize ALP via this lipid raft pathway. The reason for this uptake failure is not understood. In the present study, we show that S49AR cells are unable to synthesize SM (sphingomyelin) due to down-regulated SMS1 (SM synthase 1) expression. In parental S49 cells, resistance to ALP could be mimicked by small interfering RNA-induced SMS1 suppression, resulting in SM deficiency and blockage of raft-dependent internalization of ALP and induction of apoptosis. Similar results were obtained by treatment of the cells with myriocin/ISP-1, an inhibitor of general sphingolipid synthesis, or with U18666A, a cholesterol homoeostasis perturbing agent. U18666A is known to inhibit Niemann–Pick C1 protein-dependent vesicular transport of cholesterol from endosomal compartments to the trans-Golgi network and the plasma membrane. U18666A reduced cholesterol partitioning in detergent-resistant lipid rafts and inhibited SM synthesis in S49 cells, causing ALP resistance similar to that observed in S49AR cells. The results are explained by the strong physical interaction between (newly synthesized) SM and available cholesterol at the Golgi, where they facilitate lipid raft formation. We propose that ALP internalization by lipid-raft-dependent endocytosis represents the retrograde route of a constitutive SMS1- and lipid-raft-dependent membrane vesicular recycling process. PMID:17049047

  3. Lipid Rafts Are Physiologic Membrane Microdomains Necessary for the Morphogenic and Developmental Functions of Glial Cell Line-Derived Neurotrophic Factor In Vivo.

    PubMed

    Tsui, Cynthia C; Gabreski, Nicole A; Hein, Sarah J; Pierchala, Brian A

    2015-09-23

    Glial cell line-derived neurotrophic factor (GDNF) promotes PNS development and kidney morphogenesis via a receptor complex consisting of the glycerophosphatidylinositol (GPI)-anchored, ligand binding receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Although Ret signal transduction in vitro is augmented by translocation into lipid rafts via GFRα1, the existence and importance of lipid rafts in GDNF-Ret signaling under physiologic conditions is unresolved. A knock-in mouse was produced that replaced GFRα1 with GFRα1-TM, which contains a transmembrane (TM) domain instead of the GPI anchor. GFRα1-TM still binds GDNF and promotes Ret activation but does not translocate into rafts. In Gfrα1(TM/TM) mice, GFRα1-TM is expressed, trafficked, and processed at levels identical to GFRα1. Although Gfrα1(+/TM) mice are viable, Gfrα1(TM/TM) mice display bilateral renal agenesis, lack enteric neurons in the intestines, and have motor axon guidance deficits, similar to Gfrα1(-/-) mice. Therefore, the recruitment of Ret into lipid rafts by GFRα1 is required for the physiologic functions of GDNF in vertebrates. Significance statement: Membrane microdomains known as lipid rafts have been proposed to be unique subdomains in the plasma membrane that are critical for the signaling functions of multiple receptor complexes. Their existence and physiologic relevance has been debated. Based on in vitro studies, lipid rafts have been reported to be necessary for the function of the Glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors. The receptor for GDNF comprises the lipid raft-resident, glycerophosphatidylinositol-anchored receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Here we demonstrate, using a knock-in mouse model in which GFRα1 is no longer located in lipid rafts, that the developmental functions of GDNF in the periphery require the translocation of the GDNF receptor complex

  4. Lipid Rafts Are Physiologic Membrane Microdomains Necessary for the Morphogenic and Developmental Functions of Glial Cell Line-Derived Neurotrophic Factor In Vivo

    PubMed Central

    Tsui, Cynthia C.; Gabreski, Nicole A.; Hein, Sarah J.

    2015-01-01

    Glial cell line-derived neurotrophic factor (GDNF) promotes PNS development and kidney morphogenesis via a receptor complex consisting of the glycerophosphatidylinositol (GPI)-anchored, ligand binding receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Although Ret signal transduction in vitro is augmented by translocation into lipid rafts via GFRα1, the existence and importance of lipid rafts in GDNF–Ret signaling under physiologic conditions is unresolved. A knock-in mouse was produced that replaced GFRα1 with GFRα1–TM, which contains a transmembrane (TM) domain instead of the GPI anchor. GFRα1–TM still binds GDNF and promotes Ret activation but does not translocate into rafts. In Gfrα1TM/TM mice, GFRα1–TM is expressed, trafficked, and processed at levels identical to GFRα1. Although Gfrα1+/TM mice are viable, Gfrα1TM/TM mice display bilateral renal agenesis, lack enteric neurons in the intestines, and have motor axon guidance deficits, similar to Gfrα1−/− mice. Therefore, the recruitment of Ret into lipid rafts by GFRα1 is required for the physiologic functions of GDNF in vertebrates. SIGNIFICANCE STATEMENT Membrane microdomains known as lipid rafts have been proposed to be unique subdomains in the plasma membrane that are critical for the signaling functions of multiple receptor complexes. Their existence and physiologic relevance has been debated. Based on in vitro studies, lipid rafts have been reported to be necessary for the function of the Glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors. The receptor for GDNF comprises the lipid raft-resident, glycerophosphatidylinositol-anchored receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Here we demonstrate, using a knock-in mouse model in which GFRα1 is no longer located in lipid rafts, that the developmental functions of GDNF in the periphery require the translocation of the GDNF receptor complex

  5. Mono-ubiquitylated ORF45 Mediates Association of KSHV Particles with Internal Lipid Rafts for Viral Assembly and Egress

    PubMed Central

    Wang, Xin; Zhu, Nannan; Li, Wenwei; Zhu, Fanxiu; Wang, Yan; Yuan, Yan

    2015-01-01

    Herpesviruses acquire their envelope by budding into the lumen of cytoplasmic membrane vesicles. This process is initiated by component(s) on viral particles, which recognize the budding site where the viral glycoproteins are present and recruit cellular cargo transport and sorting machinery to the site to complete the budding process. Proteins in the tegument layer, connecting capsid and envelope, are candidates for the recognition of budding sites on vesicle membrane and induction of budding and final envelopment. We examined several outer and matrix tegument proteins of Kaposi’s sarcoma-associated herpesvirus (KSHV) and found that ORF45 associates with lipid rafts (LRs) of cellular membrane. LRs are membrane micro-domains, which have been implicated as relay stations in intracellular signaling and transport including viral entry and virion assembly. The ability of ORF45 to target LR is dependent on the mono-ubiquitylation of ORF45 at Lys297 as the mutation at Lys297 (K297R) abolished LR-association of ORF45. The K297R mutation also impairs ORF45 and viral particle co-localization with trans-Golgi network and endosomes, but facilitates ORF45 and viral particles co-localizing with lysosomes. More importantly, the recombinant KSHV carrying ORF45 K297R mutant (BAC-K297R) was found severely defective in producing mature and infectious virion particles in comparison to wild type KSHV (BAC16). Taken together, our results reveal a new function of KSHV tegument protein ORF45 in targeting LR of host cell membrane, promoting viral particles co-localization with trans-Golgi and endosome vesicles and facilitating the maturation and release of virion particles, suggesting that ORF45 plays a role in bringing KSHV particles to the budding site on cytoplasmic vesicle membrane and triggering the viral budding process for final envelopment and virion maturation. PMID:26650119

  6. Differential uPAR recruitment in caveolar-lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis.

    PubMed

    Margheri, Francesca; Papucci, Laura; Schiavone, Nicola; D'Agostino, Riccardo; Trigari, Silvana; Serratì, Simona; Laurenzana, Anna; Biagioni, Alessio; Luciani, Cristina; Chillà, Anastasia; Andreucci, Elena; Del Rosso, Tommaso; Margheri, Giancarlo; Del Rosso, Mario; Fibbi, Gabriella

    2015-01-01

    Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid-rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar-lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro-angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid-supported mobile bilayer lipid membranes with raft-like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR-GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1-enriched biomimetic membranes, were validated by identifying a pro-angiogenic activity of GM1-enriched EPCs, based on GM1-dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti-angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar-raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar-raft partitioning of uPAR, as opposed to control and GM3-challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis. PMID:25313007

  7. Differential uPAR recruitment in caveolar-lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis

    PubMed Central

    Margheri, Francesca; Papucci, Laura; Schiavone, Nicola; D'Agostino, Riccardo; Trigari, Silvana; Serratì, Simona; Laurenzana, Anna; Biagioni, Alessio; Luciani, Cristina; Chillà, Anastasia; Andreucci, Elena; Del Rosso, Tommaso; Margheri, Giancarlo; Del Rosso, Mario; Fibbi, Gabriella

    2015-01-01

    Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid-rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar-lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro-angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid-supported mobile bilayer lipid membranes with raft-like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR-GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1-enriched biomimetic membranes, were validated by identifying a pro-angiogenic activity of GM1-enriched EPCs, based on GM1-dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti-angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar-raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar-raft partitioning of uPAR, as opposed to control and GM3-challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis. PMID:25313007

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

    PubMed

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

    2014-10-01

    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 4μg oxLDL and 25µM 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

  9. 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

  10. Saikosaponin a inhibits lipopolysaccharide-oxidative stress and inflammation in Human umbilical vein endothelial cells via preventing TLR4 translocation into lipid rafts.

    PubMed

    Fu, Yunhe; Hu, Xiaoyu; Cao, Yongguo; Zhang, Zecai; Zhang, Naisheng

    2015-12-01

    Saikosaponin a (SSa), the major triterpenoid saponin derivatives from Radix bupleuri (RB), has been reported to have anti-inflammatory effects. The aim of this study was to investigate the effects of SSa on lipopolysaccharide (LPS)-induced oxidative stress and inflammatory response in human umbilical vein endothelial cells (HUVECs). HUVECs were stimulated with LPS in the presence or absence of SSa. The levels of TNF-α and IL-8 were detected by ELISA. The expression of COX-2 and iNOS, NF-κB and IκB protein were determined by Western blotting. To investigate the protective mechanisms of SSa, TLR4 expression was detected by Western blotting and membrane lipid rafts were separated by density gradient ultracentrifugation and analyzed by immunoblotting with anti-TLR4 antibody. The results showed that SSa dose-dependently inhibited the production of ROS, TNF-α, IL-8, COX-2 and iNOS in LPS-stimulated HUVECs. Western blot analysis showed that SSa suppressed LPS-induced NF-κB activation. SSa did not affect the expression of TLR4 induced by LPS. However, translocation of TLR4 into lipid rafts and oligomerization of TLR4 induce by LPS was inhibited by SSa. Furthermore, SSa disrupted the formation of lipid rafts by depleting cholesterol. Moreover, SSa activated LXRα-ABCA1 signaling pathway, which could induce cholesterol efflux from lipid rafts. Knockdown of LXRα abrogated the anti-inflammatory effects of SSa. In conclusion, the effects of SSa is associated with activating LXRα-ABCA1 signaling pathway which results in disrupting lipid rafts by depleting cholesterol and reducing translocation of TLR4 to lipid rafts and oligomerization of TLR4, thereby attenuating LPS mediated oxidative and inflammatory responses. PMID:26475038

  11. 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

  12. Dataset of differential lipid raft and global proteomes of SILAC-labeled cystic fibrosis cells upon TNF -α stimulation.

    PubMed

    Chhuon, C; Pranke, I; Borot, F; Tondelier, D; Lipecka, J; Fritsch, J; Chanson, M; Edelman, A; Ollero, M; Guerrera, I C

    2016-12-01

    Cystic fibrosis (CF) is a genetic disease due to mutations in the cystic fibrosis transmembrane regulator (CFTR), F508del-CFTR being the most frequent. Lipid raft-like microdomains (LRM) are regions of the plasma membrane that present a high cholesterol content and are insoluble to non-ionic detergents. LRM are essential functional and structural platforms that play an important role in the inflammatory response. CFTR is a known modulator of inflammation in LRM. Here we provide mass spectrometry data on the global impact of CFTR mutation and TNF-a stimulation on the LRM proteome. We used the Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) approach to quantify and identify 332 proteins in LRM upon TNF-a stimulation in CF cells and 1381 for the global proteome. We report two detailed tables containing lists of proteins obtained by mass spectrometry and the immunofluorescence validation results for one of these proteins, the G-protein coupled receptor 5A. These results are associated with the article "Changes in lipid raft proteome upon TNF-α stimulation of cystic fibrosis cells" (Chhuon et al., in press [1]). PMID:27626054

  13. Lipid rafts facilitate the interaction of PECAM-1 with the glycoprotein VI-FcR gamma-chain complex in human platelets.

    PubMed

    Lee, Fiona A; van Lier, Marjolijn; Relou, Ingrid A M; Foley, Loraine; Akkerman, Jan-Willem N; Heijnen, Harry F G; Farndale, Richard W

    2006-12-22

    Glycoprotein (GP) VI, the main signaling receptor for collagen on platelets, is expressed in complex with the FcR gamma-chain. The latter contains an immunoreceptor tyrosine-based activation motif, which becomes phosphorylated, initiating a signaling cascade leading to the rapid activation and aggregation of platelets. Previous studies have shown that signaling by immunoreceptor tyrosine-based activation motif-containing receptors is counteracted by signals from receptors with immunoreceptor tyrosine-based inhibitory motifs. Here we show, by immunoprecipitation, that the GPVI-FcR gamma-chain complex associates with the immunoreceptor tyrosine-based inhibitory motif-containing receptor, PECAM-1. In platelets stimulated with collagen-related peptide (CRP-XL), tyrosine phosphorylation of PECAM-1 precedes that of the FcR gamma-chain, implying direct regulation of the former. The GPVI-FcR gamma-chain complex and PECAM-1 were present in both lipid raft and soluble fractions in human platelets; this distribution was unaltered by activation with CRP-XL. Their association occurred in lipid rafts and was lost after lipid raft depletion using methyl-beta-cyclodextrin. We propose that lipid raft clustering facilitates the interaction of PECAM-1 with the GPVI-FcR gamma-chain complex, leading to the down-regulation of the latter. PMID:17068334

  14. 7-ketocholesterol incorporation into sphingolipid/cholesterol-enriched (lipid raft) domains is impaired by vitamin E: a specific role for alpha-tocopherol with consequences on cell death.

    PubMed

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

    2009-06-01

    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 gamma-tocopherol, the alpha-tocopherol vitamin E form was found to prevent 7-ketocholesterol-mediated apoptosis of A7R5 smooth muscle cells. To be operative, alpha-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 alpha-tocopherol resulted in the redistribution of 7-ketocholesterol out of the sphingolipid/cholesterol-enriched (lipid raft) domains. In turn, fewer amounts of alpha-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 alpha-tocopherol, but not gamma-tocopherol pretreatment. PMID:19351882

  15. Glypican-1 regulates myoblast response to HGF via Met in a lipid raft-dependent mechanism: effect on migration of skeletal muscle precursor cells

    PubMed Central

    2014-01-01

    Background Via the hepatocyte growth factor receptor (Met), hepatocyte growth factor (HGF) exerts key roles involving skeletal muscle development and regeneration. Heparan sulfate proteoglycans (HSPGs) are critical modulators of HGF activity, but the role of specific HSPGs in HGF regulation is poorly understood. Glypican-1 is the only HSPG expressed in myoblasts that localize in lipid raft membrane domains, controlling cell responses to extracellular stimuli. We determined if glypican-1 in these domains is necessary to stabilize the HGF-Met signaling complex and myoblast response to HGF. Methods C2C12 myoblasts and a derived clone (C6) with low glypican-1 expression were used as an experimental model. The activation of Met, ERK1/2 and AKT in response to HGF was evaluated. The distribution of Met and its activated form in lipid raft domains, as well as its dependence on glypican-1, were characterized by sucrose density gradient fractionation in both cell types. Rescue experiments reexpressing glypican-1 or a chimeric glypican-1 fused to the transmembrane and cytoplasmic domains of mouse syndecan-1 or myoblast pretreatment with MβCD were conducted. In vitro and in vivo myoblast migration assays in response to HGF were also performed. Results Glypican-1 localization in membrane raft domains was required for a maximum cell response to HGF. It stabilized Met and HGF in lipid raft domains, forming a signaling complex where the active phospho-Met receptor was concentrated. Glypican-1 also stabilized CD44 in a HGF-dependent manner. In addition, glypican-1 was required for in vitro and in vivo HGF-dependent myoblast migration. Conclusions Glypican-1 is a regulator of HGF-dependent signaling via Met in lipid raft domains. PMID:24517345

  16. Enhancing Protein Stability by Adsorption onto Raft-like Lipid Domains

    PubMed Central

    Litt, Jeffrey; Padala, Chakradhar; Asuri, Prashanth; Vutukuru, Srinavya; Athmakuri, Krishna; Kumar, Sanat; Dordick, Jonathan; Kane, Ravi S.

    2015-01-01

    We demonstrate that the stability of adsorbed proteins can be enhanced by controlling the heterogeneity of the surface – by creating raft-like domains in a soft liposomal membrane. Recent work has shown that enzymes adsorbed onto highly curved nanoscale supports can be more stable than those adsorbed on flat surfaces with nominally the same chemical structure. This effect has been attributed to a decrease in lateral inter-enzyme interactions on a curved surface. Exploiting this idea, we asked if adsorbing enzymes onto “patchy” surfaces composed of adsorbing and non-adsorbing regions can be used to reduce lateral interactions even on relatively flat surfaces. We demonstrate that creating domains on which an enzyme can adsorb enhances the stability of that enzyme under denaturing conditions. Furthermore, we demonstrate that the size of these domains has a considerable effect on the degree of stability imparted by adsorption. Such biomimetic raft-inspired systems may find use in applications ranging from biorecognition to the design of novel strategies for the separation of biomolecules, and controlling the interaction of multi-component membrane-bound enzymes. PMID:19385631

  17. 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. PMID:27066979

  18. Modulation of cell surface transport and lipid raft localization by the cytoplasmic tail of the influenza virus hemagglutinin.

    PubMed

    Scolari, Silvia; Imkeller, Katharina; Jolmes, Fabian; Veit, Michael; Herrmann, Andreas; Schwarzer, Roland

    2016-01-01

    Viral glycoproteins are highly variable in their primary structure, but on the other hand feature a high functional conservation to fulfil their versatile tasks during the pathogenic life cycle. Typically, all protein domains are optimized in that indispensable functions can be assigned to small conserved motifs or even individual amino acids. The cytoplasmic tail of many viral spike proteins, although of particular relevance for the virus biology, is often only insufficiently characterized. Hemagglutinin (HA), the receptor-binding protein of the influenza virus comprises a short cytoplasmic tail of 13 amino acids that exhibits three highly conserved palmitoylation sites. However, the particular importance of these modifications and the tail in general for intracellular trafficking and lateral membrane organization remains elusive. In this study, we generated HA core proteins consisting of transmembrane domain, cytoplasmic tail and a minor part of the ectodomain, tagged with a yellow fluorescent protein. Different mutation and truncation variants of these chimeric proteins were investigated using confocal microscopy, to characterize the role of cytoplasmic tail and palmitoylation for the intracellular trafficking to plasma membrane and Golgi apparatus. In addition, we assessed raft partitioning of the variants by Foerster resonance energy transfer with an established raft marker. We revealed a substantial influence of the cytoplasmic tail length on the intracellular distribution and surface exposure of the proteins. A complete removal of the tail hampers a physiological trafficking of the protein, whereas a partial truncation can be compensated by cytoplasmic palmitoylations. Plasma membrane raft partitioning on the other hand was found to imperatively require palmitoylations, and the cysteine at position 551 turned out to be of most relevance. Our data shed further light on the tight interconnection between cytoplasmic elements and intracellular trafficking and

  19. A new approach to comparing anti-CD20 antibodies: importance of the lipid rafts in their lytic efficiency

    PubMed Central

    Hammadi, Mariam; Pers, Jacques-Olivier; Berthou, Christian; Youinou, Pierre; Bordron, Anne

    2010-01-01

    The view that B lymphocytes are pathogenic in diverse pathological settings is supported by the efficacy of B-cell-ablative therapy in lymphoproliferative disorders, autoimmune diseases and graft rejection. Anti-B-cell antibodies (Abs) directed against CD20 have therefore been generated, and of these, rituximab was the first anti-CD20 monoclonal Ab (mAb) to be applied. Rituximab-mediated apoptosis, complement-dependent cytotoxicity and Ab-dependent cellular cytotoxicity differ from one disease to another, and, for the same disease, from one patient to another. This knowledge has prompted the development of new anti-CD20 mAbs in the hope of improving B-cell depletion. The inclusion of CD20/anti-CD20 complexes in large lipid rafts (LRs) enhances the results of some, but not all, anti-CD20 mAbs, and it may be possible to include smaller LRs. Lipid contents of membrane may be abnormal in malignant B-cells, and could explain resistance to treatment. The function of these mAbs and the importance of LRs warrant further investigation. A detailed understanding of them will increase results for B-cell depletion in lymphoproliferative diseases. PMID:20616960

  20. 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. PMID:26085308

  1. Lipid raft facilitated ligation of K-{alpha}1-tubulin by specific antibodies on epithelial cells: Role in pathogenesis of chronic rejection following human lung transplantation

    SciTech Connect

    Tiriveedhi, Venkataswarup; Angaswamy, Nataraju; Weber, Joseph; Mohanakumar, T.

    2010-08-20

    Research highlights: {yields} Addition of KAT Abs (+) sera to NHBE culture causes upregulation of growth factors. {yields} Cholesterol depletion causes down regulation of growth factor expression. {yields} Cholesterol depletion is accompanied by loss of membrane bound caveolin. {yields} Thus, we demonstrate lipid raft are critical for efficient ligation of the KAT Abs. -- Abstract: Long term function of human lung allografts is hindered by development of chronic rejection manifested as Bronchiolitis Obliterans Syndrome (BOS). We have previously identified the development of antibodies (Abs) following lung transplantation to K-{alpha}1-tubulin (KAT), an epithelial surface gap junction cytoskeletal protein, in patients who develop BOS. However, the biochemical and molecular basis of the interactions and signaling cascades mediated by KAT Abs are yet to be defined. In this report, we investigated the biophysical basis of the epithelial cell membrane surface interaction between KAT and its specific Abs. Towards this, we analyzed the role of the lipid raft-domains in the membrane interactions which lead to cell signaling and ultimately increased growth factor expression. Normal human bronchial epithelial (NHBE) cells, upon specific ligation with Abs to KAT obtained either from the serum of BOS(+) patients or monoclonal KAT Abs, resulted in upregulation of growth factors VEGF, PDGF, and bFGF (6.4 {+-} 1.1-, 3.2 {+-} 0.9-, and 3.4 {+-} 1.1-fold increase, respectively) all of which are important in the pathogenesis of BOS. To define the role for lipid raft in augmenting surface interactions, we analyzed the changes in the growth factor expression pattern upon depletion and enrichment with lipid raft following the ligation of the epithelial cell membranes with Abs specific for KAT. NHBE cells cultured in the presence of {beta}-methyl cyclodextran ({beta}MCD) had significantly reduced growth factor expression (1.3 {+-} 0.3, vs {beta}MCD untreated being 6.4 {+-} 1.1-fold

  2. Neuropeptide FF-sensitive confinement of mu opioid receptor does not involve lipid rafts in SH-SY5Y cells

    SciTech Connect

    Mouledous, Lionel

    2008-08-15

    *: Mu opioid (MOP) receptor activation can be functionally modulated by stimulation of Neuropeptide FF 2 (NPFF{sub 2}) G protein-coupled receptors. Fluorescence recovery after photobleaching experiments have shown that activation of the NPFF{sub 2} receptor dramatically reduces the fraction of MOP receptors confined in microdomains of the plasma membrane of SH-SY5Y neuroblastoma cells. The aim of the present work was to assess if the direct observation of receptor compartmentation by fluorescence techniques in living cells could be related to indirect estimation of receptor partitioning in lipid rafts after biochemical fractionation of the cell. Our results show that MOP receptor distribution in lipid rafts is highly dependent upon the method of purification, questioning the interpretation of previous data regarding MOP receptor compartmentation. Moreover, the NPFF analogue 1DMe does not modify the distribution profile of MOP receptors, clearly demonstrating that membrane fractionation data do not correlate with direct measurement of receptor compartmentation in living cells.

  3. Mechanism of PKA-dependent and lipid-raft independent stimulation of Connexin43 expression by oxytoxin in mouse embryonic stem cells.

    PubMed

    Yun, Seung Pil; Park, Su Shin; Ryu, Jung Min; Park, Jae Hong; Kim, Mi Ok; Lee, Jang-Hern; Han, Ho Jae

    2012-07-01

    Previous studies shows that connexins appear very early during murine embryo development, the gap junctional intercellular communication found in the inner cell mass of early embryo is also maintained in embryonic stem cells (ESC), and expression of oxytocin receptor (OTR) is developmentally regulated at early embryonic development. However, effect of oxytocin (OT) on the regulation of the connexin43 (Cx43) and maintenance of undifferentiation is not fully understood in stem cells. Therefore, we investigated the effect of OT on Cx43 expression and related signaling cascades in mouse ESC. OT increased Cx43 expression that was inhibited by the OTR inhibitor atosiban. In experiments to examine whether the effect of OT depends on lipid rafts, caveolin-1 (cav-1), cav-2, and flotillin-2, but not OTR, were detected in lipid raft fractions. Also, colocalization of OTR, cav-1, and cav-2 was not detected. Moreover, the lipid raft disruptor methyl-β-cyclodextrin did not attenuate OT-induced Cx43 expression. In experiments to examine related signaling pathways, OT activated cAMP/protein kinase A (PKA) which was inhibited by adenylyl cyclase inhibitor SQ 22536 and PKA inhibitor PKI. OT increased nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) phosphorylation which was inhibited by PKI. OT also increased cAMP response element-binding (CREB)/CREB-binding protein (CBP) expression in the nucleus and induced the formation of CREB1/NF-κB/CBP complexes, which was blocked by the NF-κB-specific small interfering RNA, NF-κB inhibitors, SN50, and bay11-7082. Complex disruption by NF-κB inhibitors decreased OT-induced Cx43 expression. In conclusion, OT stimulates Cx43 expression through the NF-κB/CREB/CBP complex via the lipid raft-independent OTR-mediated cAMP/PKA in mouse ESC. PMID:22564436

  4. Cyanidin-3-O-β-glucoside ameliorates lipopolysaccharide-induced acute lung injury by reducing TLR4 recruitment into lipid rafts.

    PubMed

    Fu, Yunhe; Zhou, Ershun; Wei, Zhengkai; Wang, Wei; Wang, Tiancheng; Yang, Zhengtao; Zhang, Naisheng

    2014-07-15

    Cyanidin-3-O-β-glucoside (C3G), a typical anthocyanin pigment that exists in the human diet, has been reported to have anti-inflammatory properties. The aim of this study was to detect the effect of C3G on LPS-induced acute lung injury and to investigate the molecular mechanisms. Acute lung injury was induced by intratracheal administration of LPS in mice. Alveolar macrophages from mice were stimulated with LPS and were treated with C3G. Our results showed that C3G attenuated lung histopathologic changes, myeloperoxidase (MPO) activity, TNF-α, IL-1β and IL-6 production in LPS-induced acute lung injury model. In vitro, C3G dose-dependently inhibited TNF-α, IL-1β, IL-6, IL-10 and IFN-β production, as well as NF-κB and IRF3 activation in LPS-stimulated alveolar macrophages. Furthermore, C3G disrupted the formation of lipid rafts by depleting cholesterol and inhibited TLR4 translocation into lipid rafts. Moreover, C3G activated LXRα-ABCG1-dependent cholesterol efflux. Knockout of LXRα abrogated the anti-inflammatory effects of C3G. In conclusion, C3G has a protective effect on LPS-induced acute lung injury. The promising anti-inflammatory mechanisms of C3G is associated with up-regulation of the LXRα-ABCG1 pathway which result in disrupting lipid rafts by depleting cholesterol and reducing translocation of TLR4 to lipid rafts, thereby suppressing TLR4 mediated inflammatory response. PMID:24841888

  5. Fluorescence-based evaluation of the partitioning of lipids and lipidated peptides into liquid-ordered lipid microdomains: a model for molecular partitioning into "lipid rafts".

    PubMed Central

    Wang, T Y; Leventis, R; Silvius, J R

    2000-01-01

    A fluorescence-quenching assay is described that can directly monitor the relative extents of partitioning of different but structurally homologous fluorescent molecules into liquid-ordered (l(o)) domains in lipid vesicles exhibiting liquid-ordered/liquid-disordered (l(o)/l(d)) phase coexistence. Applying this assay to a series of bimane-labeled diacyl phospholipid probes in cholesterol-containing ternary lipid mixtures exhibiting l(o)/l(d) phase separation, we demonstrate that partitioning into l(o)-phase domains is negligible for diunsaturated species and greatest for long-chain disaturated species. These conclusions agree well with those derived from previous studies of the association of lipids and lipid-anchored molecules with l(o)-phase domains, using methods based on the isolation of a detergent-insoluble fraction from model or biological membranes at low temperatures. However, we also find that monounsaturated and shorter-chain saturated species partition into l(o) phases with significant, albeit modest affinities, and that the level of partitioning of these latter species into l(o)-phase domains is significantly underestimated (relative to that of their long-chain saturated counterparts) by the criterion of low-temperature detergent insolubility. Finally, applying the fluorescence-quenching method to a family of lipid-modified peptides, we demonstrate that the S-palmitoyl/S-isoprenyl dual-lipidation motif found in proteins such as H- and N-ras and yeast Ste18p does not promote significant association with l(o) domains in l(o)/l(d)-phase-separated bilayers. PMID:10920023

  6. Vibrio vulnificus VvhA induces autophagy-related cell death through the lipid raft-dependent c-Src/NOX signaling pathway.

    PubMed

    Song, Eun Ju; Lee, Sei-Jung; Lim, Hyeon Su; Kim, Jun Sung; Jang, Kyung Ku; Choi, Sang Ho; Han, Ho Jae

    2016-01-01

    VvhA, a virulent factor of Vibrio (V.) vulnificus, induces acute cell death in a destructive manner. Autophagy plays an important role in cell death, but the functional role of VvhA in autophagy-related cell death has not been elucidated yet. We found that rVvhA significantly increased LC3 puncta formation and autophagic flux in promoting the cell death of human intestinal epithelial Caco-2 cells. The cell death induced by rVvhA was independent of lysosomal permeabilizaton and caspase activation. rVvhA induced rapid phosphorylation of c-Src in the membrane lipid raft, which resulted in an increased interaction between lipid raft molecule caveolin-1 and NADPH oxidase (NOX) complex Rac1 for ROS production. NOX-mediated ROS signaling induced by rVvhA increased the phosphorylation of extracellular signal-regulated kinase (ERK) and eukaryotic translation initiation factor 2α (eIF2α) which are required for mRNA expression of Atg5 and Atg16L1 involved in autophagosome formation. In an in vivo model, VvhA increased autophagy activation and paracellular permeabilization in intestinal epithelium. Collectively, the results here show that VvhA plays a pivotal role in the pathogenesis and dissemination of V. vulnificus by autophagy upregulation, through the lipid raft-mediated c-Src/NOX signaling pathway and ERK/eIF2α activation. PMID:27250250

  7. Vibrio vulnificus VvhA induces autophagy-related cell death through the lipid raft-dependent c-Src/NOX signaling pathway

    PubMed Central

    Song, Eun Ju; Lee, Sei-Jung; Lim, Hyeon Su; Kim, Jun Sung; Jang, Kyung Ku; Choi, Sang Ho; Han, Ho Jae

    2016-01-01

    VvhA, a virulent factor of Vibrio (V.) vulnificus, induces acute cell death in a destructive manner. Autophagy plays an important role in cell death, but the functional role of VvhA in autophagy-related cell death has not been elucidated yet. We found that rVvhA significantly increased LC3 puncta formation and autophagic flux in promoting the cell death of human intestinal epithelial Caco-2 cells. The cell death induced by rVvhA was independent of lysosomal permeabilizaton and caspase activation. rVvhA induced rapid phosphorylation of c-Src in the membrane lipid raft, which resulted in an increased interaction between lipid raft molecule caveolin-1 and NADPH oxidase (NOX) complex Rac1 for ROS production. NOX-mediated ROS signaling induced by rVvhA increased the phosphorylation of extracellular signal-regulated kinase (ERK) and eukaryotic translation initiation factor 2α (eIF2α) which are required for mRNA expression of Atg5 and Atg16L1 involved in autophagosome formation. In an in vivo model, VvhA increased autophagy activation and paracellular permeabilization in intestinal epithelium. Collectively, the results here show that VvhA plays a pivotal role in the pathogenesis and dissemination of V. vulnificus by autophagy upregulation, through the lipid raft-mediated c-Src/NOX signaling pathway and ERK/eIF2α activation. PMID:27250250

  8. Lipid rafts enriched in monosialylGb5Cer carrying the stage-specific embryonic antigen-4 epitope are involved in development of mouse preimplantation embryos at cleavage stage

    PubMed Central

    2011-01-01

    Background Lipid rafts enriched in glycosphingolipids (GSLs), cholesterol and signaling molecules play an essential role not only for signal transduction started by ligand binding, but for intracellular events such as organization of actin, intracellular traffic and cell polarity, but their functions in cleavage division of preimplantation embryos are not well known. Results Here we show that monosialylGb5Cer (MSGb5Cer)-enriched raft domains are involved in development during the cleavage stage of mouse preimplantation embryos. MSGb5Cer preferentially localizes at the interfaces between blastomeres in mouse preimplantation embryos. Live-imaging analysis revealed that MSGb5Cer localizes in cleavage furrows during cytokinesis, and that by accumulating at the interfaces, it thickens them. Depletion of cholesterol from the cell membrane with methyl-beta-cyclodextrin (MbCD) reduced the expression of MSGb5Cer and stopped cleavage. Extensive accumulation of MSGb5Cer at the interfaces by cross-linking with anti-MSGb5Cer Mab (6E2) caused F-actin to aggregate at the interfaces and suppressed the localization of E-cadherin at the interfaces, which resulted in the cessation of cleavage. In addition, suppression of actin polymerization with cytochalasin D (CCD) decreased the accumulation of MSGb5Cer at the interfaces. In E-cadherin-targeted embryos, the MSGb5Cer-enriched raft membrane domains accumulated heterotopically. Conclusions These results indicate that MSGb5Cer-enriched raft membrane domains participate in cytokinesis in a close cooperation with the cortical actin network and the distribution of E-cadherin. PMID:21489308

  9. Lyn cooperating with lipid rafts regulates inflammatory responses in Klebsiella pneumoniae infection through the p38/NF-κB pathway

    PubMed Central

    Li, Xuefeng; Zhou, Xikun; Li, Yi; Li, Jiaxin; Privratsky, Breanna; Ye, Yan; Wu, Erxi; Gao, Hongwei; Huang, Canhua; Wu, Min

    2014-01-01

    Klebsiella pneumoniae is one of the most common pathogens in nosocomial infections and is becoming increasingly multidrug-resistant. However, the underlying molecular pathogenesis of this bacterium remains elusive, limiting the therapeutic options. Understanding the mechanism of its pathogenesis may facilitate the development of antibacterial therapeutics. Here, we show that Lyn, a pleiotropic Src tyrosine kinase, is involved in host defense against K. pneumoniae (Kp) by regulating phagocytosis process and simultaneously downregulating inflammatory responses. Using acute infection mouse models, we observed that lyn−/− mice were more susceptible to Kp with increased mortality and severe lung injury compared with wild-type mice. Kp infected-lyn−/− mice exhibited elevated inflammatory cytokines (IL-6 and TNF-α), and increased superoxide in the lung and other organs. In addition, the phosphorylation of p38 and NF-κB p65 subunit increased markedly in response to Kp infection in lyn−/− mice. We also demonstrated that the translocation of p65 from cytoplasm to nuclei increased in cultured murine lung epithelial cells by Lyn siRNA knockdown. Furthermore, lipid rafts clustered with activated Lyn and accumulated in the site of Kp invasion. Taken together, these findings revealed that Lyn may participate in host defense against Kp infection through the negative modulation of inflammatory cytokines. PMID:24338528

  10. Sphingosine Kinase 1 Localized to the Plasma Membrane Lipid Raft Microdomain Overcomes Serum Deprivation Induced Growth Inhibition

    PubMed Central

    Hengst, Jeremy A.; Francy-Guilford, Jacquelyn M.; Fox, Todd E.; Wang, Xujun; Conroy, Elizabeth J.; Yun, Jong K.

    2009-01-01

    Several studies have demonstrated that sphingosine kinase 1 (SphK1) translocates to the plasma membrane (PM) upon its activation and further suggested the plasma membrane lipid raft microdomain (PMLRM) as a target for SphK1 relocalization. To date, however, direct evidence of SphK1 localization to the PMLRM has been lacking. In this report, using multiple biochemical and subcellular fractionation techniques we demonstrate that endogenous SphK1 protein and its substrate, D-erythro sphingosine, are present within the PMLRM. Additionally, we demonstrate that the PMA stimulation of SphK1 localized to the PMLRM results in production of sphingosine-1-phosphate as well as induction of cell growth under serum-deprivation conditions. We further report that Ser225Ala and Thr54Cys mutations, reported to abrogate phosphatidylserine binding, block SphK1 targeting to the PMLRM and SphK1 induced cell growth. Together these findings provide direct evidence that the PMLRM is the major site-of-action for SphK1 to overcome serum-deprived cell growth inhibition. PMID:19782042

  11. ß1 Integrin Binding Phosphorylates Ezrin at T567 to Activate a Lipid Raft Signalsome Driving Invadopodia Activity and Invasion

    PubMed Central

    Antelmi, Ester; Cardone, Rosa A.; Greco, Maria R.; Rubino, Rosa; Di Sole, Francesca; Martino, Nicola A.; Casavola, Valeria; Carcangiu, MariaLuisa; Moro, Loredana; Reshkin, Stephan J.

    2013-01-01

    Extracellular matrix (ECM) degradation is a critical process in tumor cell invasion and requires matrix degrading protrusions called invadopodia. The Na+/H+ exchanger (NHE1) has recently been shown to be fundamental in the regulation of invadopodia actin cytoskeleton dynamics and activity. However, the structural link between the invadopodia cytoskeleton and NHE1 is still unknown. A candidate could be ezrin, a linker between the NHE1 and the actin cytoskeleton known to play a pivotal role in invasion and metastasis. However, the mechanistic basis for its role remains unknown. Here, we demonstrate that ezrin phosphorylated at T567 is highly overexpressed in the membrane of human breast tumors and positively associated with invasive growth and HER2 overexpression. Further, in the metastatic cell line, MDA-MB-231, p-ezrin was almost exclusively expressed in invadopodia lipid rafts where it co-localized in a functional complex with NHE1, EGFR, ß1-integrin and phosphorylated-NHERF1. Manipulation by mutation of ezrins T567 phosphorylation state and/or PIP2 binding capacity or of NHE1s binding to ezrin or PIP2 demonstrated that p-ezrin expression and binding to PIP2 are required for invadopodia-mediated ECM degradation and invasion and identified NHE1 as the membrane protein that p-ezrin regulates to induce invadopodia formation and activity. PMID:24086451

  12. 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

  13. Lateral diffusion of Gαs in the plasma membrane is decreased after chronic but not acute antidepressant treatment: role of lipid raft and non-raft membrane microdomains.

    PubMed

    Czysz, Andrew H; Schappi, Jeffrey M; Rasenick, Mark M

    2015-02-01

    GPCR signaling is modified both in major depressive disorder and by chronic antidepressant treatment. Endogenous Gαs redistributes from raft- to nonraft-membrane fractions after chronic antidepressant treatment. Modification of G protein anchoring may participate in this process. Regulation of Gαs signaling by antidepressants was studied using fluorescence recovery after photobleaching (FRAP) of GFP-Gαs. Here we find that extended antidepressant treatment both increases the half-time of maximum recovery of GFP-Gαs and decreases the extent of recovery. Furthermore, this effect parallels the movement of Gαs out of lipid rafts as determined by cold detergent membrane extraction with respect to both dose and duration of drug treatment. This effect was observed for several classes of compounds with antidepressant activity, whereas closely related molecules lacking antidepressant activity (eg, R-citalopram) did not produce the effect. These results are consistent with previously observed antidepressant-induced translocation of Gαs, but also suggest an alternate membrane attachment site for this G protein. Furthermore, FRAP analysis provides the possibility of a relatively high-throughput screening tool for compounds with putative antidepressant activity. PMID:25249058

  14. Lateral Diffusion of Gαs in the Plasma Membrane Is Decreased after Chronic but not Acute Antidepressant Treatment: Role of Lipid Raft and Non-Raft Membrane Microdomains

    PubMed Central

    Czysz, Andrew H; Schappi, Jeffrey M; Rasenick, Mark M

    2015-01-01

    GPCR signaling is modified both in major depressive disorder and by chronic antidepressant treatment. Endogenous Gαs redistributes from raft- to nonraft-membrane fractions after chronic antidepressant treatment. Modification of G protein anchoring may participate in this process. Regulation of Gαs signaling by antidepressants was studied using fluorescence recovery after photobleaching (FRAP) of GFP-Gαs. Here we find that extended antidepressant treatment both increases the half-time of maximum recovery of GFP-Gαs and decreases the extent of recovery. Furthermore, this effect parallels the movement of Gαs out of lipid rafts as determined by cold detergent membrane extraction with respect to both dose and duration of drug treatment. This effect was observed for several classes of compounds with antidepressant activity, whereas closely related molecules lacking antidepressant activity (eg, R-citalopram) did not produce the effect. These results are consistent with previously observed antidepressant-induced translocation of Gαs, but also suggest an alternate membrane attachment site for this G protein. Furthermore, FRAP analysis provides the possibility of a relatively high-throughput screening tool for compounds with putative antidepressant activity. PMID:25249058

  15. Depletion of phytosterols from the plant plasma membrane provides evidence for disruption of lipid rafts.

    PubMed

    Roche, Yann; Gerbeau-Pissot, Patricia; Buhot, Blandine; Thomas, Dominique; Bonneau, Laurent; Gresti, Joseph; Mongrand, Sébastien; Perrier-Cornet, Jean-Marie; Simon-Plas, Françoise

    2008-11-01

    Involvement of sterols in membrane structural properties has been extensively studied in model systems but rarely assessed in natural membranes and never investigated for the plant plasma membrane (PM). Here, we address the question of the role of phytosterols in the organization of the plant PM. The sterol composition of tobacco BY-2 cell PM was determined by gas chromatography. The cyclic oligosaccharide methyl-beta-cyclodextrin, commonly used in animal cells to decrease cholesterol levels, caused a drastic reduction (50%) in the PM total free sterol content of the plant material, without modification in amounts of steryl-conjugates. Fluorescence spectroscopy experiments using DPH, TMA-DPH, Laurdan, and di-4-ANEPPDHQ indicated that such a depletion in sterol content increased lipid acyl chain disorder and reduced the overall liquid-phase heterogeneity in correlation with the disruption of phytosterol-rich domains. Methyl-beta-cyclodextrin also prevented isolation of a PM fraction resistant to solubilization by nonionic detergents, previously characterized in tobacco, and induced redistribution of the proteic marker of this fraction, NtrbohD, within the membrane. Altogether, our results support the role of phytosterols in the lateral structuring of the PM of higher plant cells and suggest that they are key compounds for the formation of plant PM microdomains. PMID:18676403

  16. Pantethine Alters Lipid Composition and Cholesterol Content of Membrane Rafts, With Down-Regulation of CXCL12-Induced T Cell Migration.

    PubMed

    van Gijsel-Bonnello, Manuel; Acar, Niyazi; Molino, Yves; Bretillon, Lionel; Khrestchatisky, Michel; de Reggi, Max; Gharib, Bouchra

    2015-10-01

    Pantethine, a natural low-molecular-weight thiol, shows a broad activity in a large range of essential cellular pathways. It has been long known as a hypolipidemic and hypocholesterolemic agent. We have recently shown that it exerts a neuroprotective action in mouse models of cerebral malaria and Parkinson's disease through multiple mechanisms. In the present study, we looked at its effects on membrane lipid rafts that serve as platforms for molecules engaged in cell activity, therefore providing a target against inappropriate cell response leading to a chronic inflammation. We found that pantethine-treated cells showed a significant change in raft fatty acid composition and cholesterol content, with ultimate downregulation of cell adhesion, CXCL12-driven chemotaxis, and transendothelial migration of various T cell types, including human Jurkat cell line and circulating effector T cells. The mechanisms involved include the alteration of the following: (i) CXCL12 binding to its target cells; (ii) membrane dynamics of CXCR4 and CXCR7, the two CXCL12 receptors; and (iii) cell redox status, a crucial determinant in the regulation of the chemokine system. In addition, we considered the linker for activation of T cells molecule to show that pantethine effects were associated with the displacement from the rafts of the acylated signaling molecules which had their palmitoylation level reduced.. In conclusion, the results presented here, together with previously published findings, indicate that due to its pleiotropic action, pantethine can downregulate the multifaceted process leading to pathogenic T cell activation and migration. PMID:25728249

  17. Solvatochromic Nile Red probes with FRET quencher reveal lipid order heterogeneity in living and apoptotic cells.

    PubMed

    Kreder, Rémy; Pyrshev, Kyrylo A; Darwich, Zeinab; Kucherak, Oleksandr A; Mély, Yves; Klymchenko, Andrey S

    2015-06-19

    Detecting and imaging lipid microdomains (rafts) in cell membranes remain a challenge despite intensive research in the field. Two types of fluorescent probes are used for this purpose: one specifically labels a given phase (liquid ordered, Lo, or liquid disordered, Ld), while the other, being environment-sensitive (solvatochromic), stains the two phases in different emission colors. Here, we combined the two approaches by designing a phase-sensitive probe of the Ld phase and a quencher of the Ld phase. The former is an analogue of the recently developed Nile Red-based probe NR12S, bearing a bulky hydrophobic chain (bNR10S), while the latter is based on Black Hole Quencher-2 designed as bNR10S (bQ10S). Fluorescence spectroscopy of large unilamellar vesicles and microscopy of giant vesicles showed that the bNR10S probe can partition specifically into the Ld phase, while bQ10S can specifically quench the NR12S probe in the Ld phase so that only its fraction in the Lo phase remains fluorescent. Thus, the toolkit of two probes with quencher can specifically target Ld and Lo phases and identify their lipid order from the emission color. Application of this toolkit in living cells (HeLa, CHO, and 293T cell lines) revealed heterogeneity in the cell plasma membranes, observed as distinct probe environments close to the Lo and Ld phases of model membranes. In HeLa cells undergoing apoptosis, our toolkit showed the formation of separate domains of the Ld-like phase in the form of blebs. The developed tools open new possibilities in lipid raft research. PMID:25710589

  18. Altering Hydrophobic Sequence Lengths Shows That Hydrophobic Mismatch Controls Affinity for Ordered Lipid Domains (Rafts) in the Multitransmembrane Strand Protein Perfringolysin O*

    PubMed Central

    Lin, Qingqing; London, Erwin

    2013-01-01

    The hypothesis that mismatch between transmembrane (TM) length and bilayer width controls TM protein affinity for ordered lipid domains (rafts) was tested using perfringolysin O (PFO), a pore-forming cholesterol-dependent cytolysin. PFO forms a multimeric barrel with many TM segments. The properties of PFO mutants with lengthened or shortened TM segments were compared with that of PFO with wild type TM sequences. Both mutant and wild type length PFO exhibited cholesterol-dependent membrane insertion. Maximal PFO-induced pore formation occurred in vesicles with wider bilayers for lengthened TM segments and in thinner bilayers for shortened TM segments. In diC18:0 phosphatidylcholine (PC)/diC14:1 PC/cholesterol vesicles, which form ordered domains with a relatively thick bilayer and disordered domains with a relatively thin bilayer, affinity for ordered domains was greatest with lengthened TM segments and least with shortened TM segments as judged by FRET. Similar results were observed by microscopy in giant vesicles containing sphingomyelin in place of diC18:0 PC. In contrast, in diC16:0 PC/diC14:0 PC/diC20:1 PC/cholesterol vesicles, which should form ordered domains with a relatively thin bilayer and disordered domains with a relatively thick bilayer, relative affinity for ordered domains was greatest with shortened TM segments and least with lengthened TM segments. The inability of multi-TM segment proteins (unlike single TM segment proteins) to adapt to mismatch by tilting may explain the sensitivity of raft affinity to mismatch. The difference in width sensitivity for single and multi-TM helix proteins may link raft affinity to multimeric state and thus control the assembly of multimeric TM complexes in rafts. PMID:23150664

  19. Lovastatin enhances adenovirus-mediated TRAIL induced apoptosis by depleting cholesterol of lipid rafts and affecting CAR and death receptor expression of prostate cancer cells.

    PubMed

    Liu, Youhong; Chen, Lin; Gong, Zhicheng; Shen, Liangfang; Kao, Chinghai; Hock, Janet M; Sun, Lunquan; Li, Xiong

    2015-02-20

    Oncolytic adenovirus and apoptosis inducer TRAIL are promising cancer therapies. Their antitumor efficacy, when used as single agents, is limited. Oncolytic adenoviruses have low infection activity, and cancer cells develop resistance to TRAIL-induced apoptosis. Here, we explored combining prostate-restricted replication competent adenovirus-mediated TRAIL (PRRA-TRAIL) with lovastatin, a commonly used cholesterol-lowering drug, as a potential therapy for advanced prostate cancer (PCa). Lovastatin significantly enhanced the efficacy of PRRA-TRAIL by promoting the in vivo tumor suppression, and the in vitro cell killing and apoptosis induction, via integration of multiple molecular mechanisms. Lovastatin enhanced PRRA replication and virus-delivered transgene expression by increasing the expression levels of CAR and integrins, which are critical for adenovirus 5 binding and internalization. Lovastatin enhanced TRAIL-induced apoptosis by increasing death receptor DR4 expression. These multiple effects of lovastatin on CAR, integrins and DR4 expression were closely associated with cholesterol-depletion in lipid rafts. These studies, for the first time, show correlations between cholesterol/lipid rafts, oncolytic adenovirus infection efficiency and the antitumor efficacy of TRAIL at the cellular level. This work enhances our understanding of the molecular mechanisms that support use of lovastatin, in combination with PRRA-TRAIL, as a candidate strategy to treat human refractory prostate cancer in the future. PMID:25605010

  20. 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

  1. Vibrio vulnificus VvpE inhibits mucin 2 expression by hypermethylation via lipid raft-mediated ROS signaling in intestinal epithelial cells

    PubMed Central

    Lee, S-J; Jung, Y H; Oh, S Y; Jang, K K; Lee, H S; Choi, S H; Han, H J

    2015-01-01

    Mucin is an important physical barrier against enteric pathogens. VvpE is an elastase encoded by Gram-negative bacterium Vibrio vulnificus; however, the functional role of VvpE in intestinal mucin (Muc) production is yet to be elucidated. The recombinant protein (r) VvpE significantly reduced the level of Muc2 in human mucus-secreting HT29-MTX cells. The repression of Muc2 induced by rVvpE was highly susceptible to the knockdown of intelectin-1b (ITLN) and sequestration of cholesterol by methyl-β-cyclodextrin. We found that rVvpE induces the recruitment of NADPH oxidase 2 and neutrophil cytosolic factor 1 into the membrane lipid rafts coupled with ITLN to facilitate the production of reactive oxygen species (ROS). The bacterial signaling of rVvpE through ROS production is uniquely mediated by the phosphorylation of ERK, which was downregulated by the silencing of the PKCδ. Moreover, rVvpE induced region-specific methylation in the Muc2 promoter to promote the transcriptional repression of Muc2. In two mouse models of V. vulnificus infection, the mutation of the vvpE gene from V. vulnificus exhibited an increased survival rate and maintained the level of Muc2 expression in intestine. These results demonstrate that VvpE inhibits Muc2 expression by hypermethylation via lipid raft-mediated ROS signaling in the intestinal epithelial cells. PMID:26086960

  2. NADPH oxidase and lipid raft-associated redox signaling are required for PCB153-induced upregulation of cell adhesion molecules in human brain endothelial cells.

    PubMed

    Eum, Sung Yong; Andras, Ibolya; Hennig, Bernhard; Toborek, Michal

    2009-10-15

    Exposure to persistent organic pollutants, such as polychlorinated biphenyls (PCBs), can lead to chronic inflammation and the development of vascular diseases. Because cell adhesion molecules (CAMs) of the cerebrovascular endothelium regulate infiltration of inflammatory cells into the brain, we have explored the molecular mechanisms by which ortho-substituted polychlorinated biphenyls (PCBs), such as PCB153, can upregulate CAMs in brain endothelial cells. Exposure to PCB153 increased expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), as well as elevated adhesion of leukocytes to brain endothelial cells. These effects were impeded by inhibitors of EGFR, JAKs, or Src activity. In addition, pharmacological inhibition of NADPH oxidase or disruption of lipid rafts by cholesterol depleting agents blocked PCB153-induced phosphorylation of JAK and Src kinases and upregulation of CAMs. In contrast, silencing of caveolin-1 by siRNA interference did not affect upregulation of ICAM-1 and VCAM-1 in brain endothelial cells stimulated by PCB153. Results of the present study indicate that lipid raft-dependent NADPH oxidase/JAK/EGFR signaling mechanisms regulate the expression of CAMs in brain endothelial cells and adhesion of leukocytes to endothelial monolayers. Due to its role in leukocyte infiltration, induction of CAMs may contribute to PCB-induced cerebrovascular disorders and neurotoxic effects in the CNS. PMID:19632255

  3. NADPH oxidase and lipid raft-associated redox signaling are required for PCB153-induced upregulation of cell adhesion molecules in human brain endothelial cells

    SciTech Connect

    Eum, Sung Yong Andras, Ibolya; Hennig, Bernhard; Toborek, Michal

    2009-10-15

    Exposure to persistent organic pollutants, such as polychlorinated biphenyls (PCBs), can lead to chronic inflammation and the development of vascular diseases. Because cell adhesion molecules (CAMs) of the cerebrovascular endothelium regulate infiltration of inflammatory cells into the brain, we have explored the molecular mechanisms by which ortho-substituted polychlorinated biphenyls (PCBs), such as PCB153, can upregulate CAMs in brain endothelial cells. Exposure to PCB153 increased expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), as well as elevated adhesion of leukocytes to brain endothelial cells. These effects were impeded by inhibitors of EGFR, JAKs, or Src activity. In addition, pharmacological inhibition of NADPH oxidase or disruption of lipid rafts by cholesterol depleting agents blocked PCB153-induced phosphorylation of JAK and Src kinases and upregulation of CAMs. In contrast, silencing of caveolin-1 by siRNA interference did not affect upregulation of ICAM-1 and VCAM-1 in brain endothelial cells stimulated by PCB153. Results of the present study indicate that lipid raft-dependent NADPH oxidase/JAK/EGFR signaling mechanisms regulate the expression of CAMs in brain endothelial cells and adhesion of leukocytes to endothelial monolayers. Due to its role in leukocyte infiltration, induction of CAMs may contribute to PCB-induced cerebrovascular disorders and neurotoxic effects in the CNS.

  4. The binding of Aβ1-42 to lipid rafts of RBC is enhanced by dietary docosahexaenoic acid in rats: Implicates to Alzheimer's disease.

    PubMed

    Hashimoto, Michio; Hossain, Shahdat; Katakura, Masanori; Al Mamun, Abdullah; Shido, Osamu

    2015-06-01

    Once amyloid β peptides (Aβs) of the Alzheimer's disease build up in blood circulation, they are capable of binding to red blood cell (RBC) and inducing hemolysis of RBC. The mechanisms of the interactions between RBC and Aβ are largely unknown; however, it is very important for the therapeutic target of Aβ-induced hemolysis. In the present study, we investigated whether Aβ1-42 interacts with caveolin-1-containing detergent-resistant membranes (DRMs) of RBC and whether the interaction could be modulated by dietary pre-administration of docosahexaenoic acid (DHA). DHA pre-administration to rats inhibited hemolysis by Aβ1-42. This activity was accompanied by increased DHA levels and membrane fluidity and decreased cholesterol level, lipid peroxidation, and reactive oxygen species in the RBCs of the DHA-pretreated rats, suggesting that the antioxidative property of DHA may rescue RBCs from oxidative damage by Aβ1-42. The level of caveolin-1 was augmented in the DRMs of DHA-pretreated rats. Binding between Aβ1-42 and DRMs of RBC significantly increased in DHA-rats. When fluorescently labeled Aβ1-42 (TAMRA-Aβ1-42) was directly infused into the bloodstream, it again occupied the caveolin-1-containing DRMs of the RBCs from the DHA-rats to a greater extent, indicating that circulating Aβs interact with the caveolin-1-rich lipid rafts of DRMs and the interaction is stronger in the DHA-enriched RBCs. The levels of TAMRA-Aβ1-42 also increased in liver DRMs, whereas it decreased in plasma of DHA-pretreated rats. DHA might help clearance of circulating Aβs by increased lipid raft-dependent degradation pathways and implicate to therapies in Alzheimer's disease. PMID:25782726

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

    PubMed

    Nomura, Sayaka; Iwata, Satoshi; Hatano, Ryo; Komiya, Eriko; Dang, Nam H; Iwao, Noriaki; Ohnuma, Kei; Morimoto, Chikao

    2016-05-20

    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 regulating 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. PMID:27103437

  6. Human Immunodeficiency Virus Type 1 Enters Brain Microvascular Endothelia by Macropinocytosis Dependent on Lipid Rafts and the Mitogen-Activated Protein Kinase Signaling Pathway

    PubMed Central

    Liu, Nancy Q.; Lossinsky, Albert S.; Popik, Waldemar; Li, Xia; Gujuluva, Chandrasekhar; Kriederman, Benjamin; Roberts, Jaclyn; Pushkarsky, Tatania; Bukrinsky, Michael; Witte, Marlys; Weinand, Martin; Fiala, Milan

    2002-01-01

    Brain microvascular endothelial cells (BMVECs) present an incomplete barrier to human immunodeficiency virus type 1 (HIV-1) neuroinvasion. In order to clarify the mechanisms of HIV-1 invasion, we have examined HIV-1 uptake and transcellular penetration in an in vitro BMVEC model. No evidence of productive infection was observed by luciferase, PCR, and reverse transcriptase assays. Approximately 1% of viral RNA and 1% of infectious virus penetrated the BMVEC barrier without disruption of tight junctions. The virus upregulated ICAM-1 on plasma membranes and in cytoplasmic vesiculotubular structures. HIV-1 virions were entangled by microvilli and were taken into cytoplasmic vesicles through surface invaginations without fusion of the virus envelope with the plasma membrane. Subsequently, the cytoplasmic vesicles fused with lysosomes, the virions were lysed, and the vesicles diminished in size. Upon cell entry, HIV-1 colocalized with cholera toxin B, which targets lipid raft-associated GM1 ganglioside. Cholesterol-extracting agents, cyclodextrin and nystatin, and polyanion heparin significantly inhibited virus entry. Anti-CD4 had no effect and the chemokine AOP-RANTES had only a slight inhibitory effect on virus entry. HIV-1 activated the mitogen-activated protein kinase (MAPK) pathway, and inhibition of MAPK/Erk kinase inhibited virus entry. Entry was also blocked by dimethylamiloride, indicating that HIV-1 enters endothelial cells by macropinocytosis. Therefore, HIV-1 penetrates BMVECs in ICAM-1-lined macropinosomes by a mechanism involving lipid rafts, MAPK signaling, and glycosylaminoglycans, while CD4 and chemokine receptors play limited roles in this process. PMID:12050382

  7. Linker for Activation of T-cell Family Member2 (LAT2) a Lipid Raft Adaptor Protein for AKT Signaling, Is an Early Mediator of Alkylphospholipid Anti-leukemic Activity*

    PubMed Central

    Thomé, Carolina H.; dos Santos, Guilherme A.; Ferreira, Germano A.; Scheucher, Priscila S.; Izumi, Clarice; Leopoldino, Andreia M.; Simão, Ana Maria; Ciancaglini, Pietro; de Oliveira, Kleber T.; Chin, Alice; Hanash, Samir M.; Falcão, Roberto P.; Rego, Eduardo M.; Greene, Lewis J.; Faça, Vitor M.

    2012-01-01

    Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 μm 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 μm ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five μm ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5×), perifosine (3×), and arsenic trioxide (8.5×). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy. PMID:23001822

  8. Linker for activation of T-cell family member2 (LAT2) a lipid raft adaptor protein for AKT signaling, is an early mediator of alkylphospholipid anti-leukemic activity.

    PubMed

    Thomé, Carolina H; dos Santos, Guilherme A; Ferreira, Germano A; Scheucher, Priscila S; Izumi, Clarice; Leopoldino, Andreia M; Simão, Ana Maria; Ciancaglini, Pietro; de Oliveira, Kleber T; Chin, Alice; Hanash, Samir M; Falcão, Roberto P; Rego, Eduardo M; Greene, Lewis J; Faça, Vitor M

    2012-12-01

    Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 μm 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 μm ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five μm ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5×), perifosine (3×), and arsenic trioxide (8.5×). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy. PMID:23001822

  9. Regulation of Renal Organic Anion Transporter 3 (SLC22A8) Expression and Function by the Integrity of Lipid Raft Domains and their Associated Cytoskeleton

    PubMed Central

    Srimaroeng, Chutima; Cecile, Jennifer Perry; Walden, Ramsey; Pritchard, John B.

    2013-01-01

    Background/Aims In humans and rodents, organic anion transporter 3 (Oat3) is highly expressed on the basolateral membrane of renal proximal tubules and mediates the secretion of exogenous and endogenous anions. Regulation of Oat3 expression and function has been observed in both expression system and intact renal epithelia. However, information on the local membrane environment of Oat3 and its role is limited. Lipid raft domains (LRD; cholesterol-rich domains of the plasma membrane) play important roles in membrane protein expression, function and targeting. In the present study, we have examined the role of LRD-rich membranes and their associated cytoskeletal proteins on Oat3 expression and function. Methods LRD-rich membranes were isolated from rat renal cortical tissues and from HEK-293 cells stably expressing human OAT3 (hOAT3) by differential centrifugation with triton X-100 extraction. Western blots were subsequently analyzed to determine protein expression. In addition, the effect of disruption of LRD-rich membranes was examined on functional Oat3 mediated estrone sulfate (ES) transport in rat renal cortical slices. Cytoskeleton disruptors were investigated in both hOAT3 expressing HEK-293 cells and rat renal cortical slices. Results Lipid-enriched membranes from rat renal cortical tissues and hOAT3-expressing HEK-293 cells showed co-expression of rOat3/hOAT3 and several lipid raft-associated proteins, specifically caveolin 1 (Cav1), β-actin and myosin. Moreover, immunohistochemistry in hOAT3-expressing HEK-293 cells demonstrated that these LRD-rich proteins co-localized with hOAT3. Potassium iodide (KI), an inhibitor of protein-cytoskeletal interaction, effectively detached cytoskeleton proteins and hOAT3 from plasma membrane, leading to redistribution of hOAT3 into non-LRD-rich compartments. In addition, inhibition of cytoskeleton integrity and membrane trafficking processes significantly reduced ES uptake mediated by both human and rat Oat3. Cholesterol

  10. Rafts, little caves and large potholes: how lipid structure interacts with membrane proteins to create functionally diverse membrane environments.

    PubMed

    Morris, Roger; Cox, Helen; Mombelli, Enrico; Quinn, Peter J

    2004-01-01

    This chapter reviews how diverse lipid microdomains form in the membrane and partition proteins into different functional units that regulate cell trafficking, signalling and movement. We will concentrate upon five major issues: 1. the diversity of lipid structure that produces diverse microenvironments into which different subsets of proteins partition; 2. why ordered lipid domains exclude proteins, and the conditions required for select subsets of proteins to enter these domains; 3. the coupling of the inner and outer leaflets within ordered microdomains; 4. the effect of ordered lipid domains upon membrane properties including curvature and hydrophobicity that affect membrane fission, fusion and extension of filopodia; 5. the biological effects of these structural constraints; in particular how the properties of these domains combine to provide a very different signalling, trafficking and membrane fusion environment to that found in disordered (fluid mosaic) membrane. In addressing these problems, the review draws upon studies ranging from molecular dynamic modelling of lipid interactions, through physical studies of model membrane systems to structural and biological studies of whole cells, examining in the process problems inherent in visualising and purifying these microdomains. While the diversity of structure and function of ordered lipid microdomains is emphasised, some general roles emerge. In particular, the basis for having quite different, non-interacting ordered lipid domains on the same membrane is evident in the diversity of lipid structure and plays a key role in sorting signalling systems. The exclusion of ordered membrane from coated pits, and hence rapid endocytosis, is suggested to underlie the ability of highly ordered domains to establish stable secondary signalling systems required, for instance, in T cell receptor, insulin and neurotrophin signalling. PMID:15376618

  11. Energetic modeling and single-molecule verification of dynamic regulation on receptor complexes by actin corrals and lipid raft domains

    NASA Astrophysics Data System (ADS)

    Lin, Chien Y.; Huang, Jung Y.; Lo, Leu-Wei

    2014-12-01

    We developed an energetic model by integrating the generalized Langevin equation with the Cahn-Hilliard equation to simulate the diffusive behaviors of receptor proteins in the plasma membrane of a living cell. Simulation results are presented to elaborate the confinement effects from actin corrals and protein-induced lipid domains. Single-molecule tracking data of epidermal growth factor receptors (EGFR) acquired on live HeLa cells agree with the simulation results and the mechanism that controls the diffusion of single-molecule receptors is clarified. We discovered that after ligand binding, EGFR molecules move into lipid nanodomains. The transition rates between different diffusion states of liganded EGFR molecules are regulated by the lipid domains. Our method successfully captures dynamic interactions of receptors at the single-molecule level and provides insight into the functional architecture of both the diffusing EGFR molecules and their local cellular environment.

  12. 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

    SciTech Connect

    Du, Yijun; Pattnaik, Asit K.; Song, Cheng; Yoo, Dongwan; Li, Gang

    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 that 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.

  13. 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 Central

    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

    2015-01-01

    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 Ca2+ 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. PMID:25605290

  14. CR3 and Dectin-1 Collaborate in Macrophage Cytokine Response through Association on Lipid Rafts and Activation of Syk-JNK-AP-1 Pathway.

    PubMed

    Huang, Juin-Hua; Lin, Ching-Yu; Wu, Sheng-Yang; Chen, Wen-Yu; Chu, Ching-Liang; Brown, Gordon D; Chuu, Chih-Pin; Wu-Hsieh, Betty A

    2015-07-01

    Collaboration between heterogeneous pattern recognition receptors (PRRs) leading to synergistic coordination of immune response is important for the host to fight against invading pathogens. Although complement receptor 3 (CR3) and Dectin-1 are major PRRs to detect fungi, crosstalk between these two receptors in antifungal immunity is largely undefined. Here we took advantage of Histoplasma capsulatum which is known to interact with both CR3 and Dectin-1 and specific particulate ligands to study the collaboration of CR3 and Dectin-1 in macrophage cytokine response. By employing Micro-Western Array (MWA), genetic approach, and pharmacological inhibitors, we demonstrated that CR3 and Dectin-1 act collaboratively to trigger macrophage TNF and IL-6 response through signaling integration at Syk kinase, allowing subsequent enhanced activation of Syk-JNK-AP-1 pathway. Upon engagement, CR3 and Dectin-1 colocalize and form clusters on lipid raft microdomains which serve as a platform facilitating their cooperation in signaling activation and cytokine production. Furthermore, in vivo studies showed that CR3 and Dectin-1 cooperatively participate in host defense against disseminated histoplasmosis and instruct adaptive immune response. Taken together, our findings define the mechanism of receptor crosstalk between CR3 and Dectin-1 and demonstrate the importance of their collaboration in host defense against fungal infection. PMID:26132276

  15. 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

  16. Neohesperidin dihydrochalcone down-regulates MyD88-dependent and -independent signaling by inhibiting endotoxin-induced trafficking of TLR4 to lipid rafts.

    PubMed

    Xia, Xiaomin; Fu, Juanli; Song, Xiufang; Shi, Qiong; Su, Chuanyang; Song, Erqun; Song, Yang

    2015-12-01

    Fulminant hepatic failure (FHF) is a lethal clinical syndrome characterized by the activation of macrophages and the increased production of inflammatory mediators. The purpose of this study was to investigate the effects of neohesperidin dihydrochalcone (NHDC), a widely-used low caloric artificial sweetener against FHF. An FHF experimental model was established in mice by intraperitoneal injection of D-galactosamine (d-GalN) (400mg/kg)/lipopolysaccharides (LPS) (10 μg/kg). Mice were orally administered NHDC for 6 continuous days and at 1h before d-GalN/LPS administration. RAW264.7 macrophages were used as an in vitro model. Cells were pre-treated with NHDC for 1h before stimulation with LPS (10 μg/ml) for 6h. d-GalN/LPS markedly increased the serum transaminase activities and levels of oxidative and inflammatory markers, which were significantly attenuated by NHDC. Mechanistic analysis indicated that NHDC inhibited LPS-induced myeloid differentiation factor 88 (MyD88) and TIR-containing adapter molecule (TRIF)-dependent signaling. Transient transfection of TLR4 or MyD88 siRNA inhibited the downstream inflammatory signaling. This effect could also be achieved by the pretreatment with NHDC. The fluorescence microscopy and flow cytometry results suggested that NHDC potently inhibited the binding of LPS to TLR4 in RAW264.7 macrophages. In addition, the inhibitory effect of NHDC on LPS-induced translocation of TLR4 into lipid raft domains played an important role in the amelioration of production of downstream pro-inflammatory molecules. Furthermore, the activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) by NHDC inhibited TLR4 signaling. In conclusion, our results suggest that NHDC attenuates d-GalN/LPS-induced FHF by inhibiting the TLR4-mediated inflammatory pathway, demonstrating a new application of NHDC as a hepatoprotective agent. PMID:26453923

  17. A New Membrane Lipid Raft Gene SpFLT-1 Facilitating the Endocytosis of Vibrio alginolyticus in the Crab Scylla paramamosain

    PubMed Central

    Chen, Fangyi; Bo, Jun; Ma, Xiaowan; Dong, Lixia; Shan, Zhongguo; Cui, Qian; Chen, Huiyun; Wang, Kejian

    2015-01-01

    Pathogens can enter their host cells by way of endocytosis in which the membrane lipid raft gene flotillins are probably involved in the invasion process and this is an important way to cause infection. In this study, a new gene SpFLT-1 was identified in Scylla paramamosain, which shared high identity with the flotillin-1 of other species. The SpFLT-1 gene was widely distributed in tissues and showed the highest level of mRNA transcripts in the hemocytes. This gene might be a maternal gene based on the evident results that it was highly expressed in maternal ovaries and in the early developmental stages of the zygote and early embryo stage whereas it gradually decreased in zoea 1. SpFLT-1 positively responded to the challenge of Vibrio alginolyticus with a significantly increased level of mRNA expression in the hemocytes and gills at 3 hours post infection (hpi). The SpFLT-1 protein was detected densely in the same fraction layer where the Vibrio protein was most present in the hemocytes and gills at 3 hpi. Furthermore, it was found that the expression of SpFLT-1 decreased to the base level following disappearance of the Vibrio protein at 6 hpi in the gills. Silencing SpFLT-1 inhibited the endocytosis rate of V. alginolyticus but overexpression of the gene could facilitate bacterial entry into the epithelioma papulosum cyprinid cells. Our study indicated that SpFLT-1 may act as a key protein involved in the process of bacterial infection and this sheds light on clarifying the pathogenesis of pathogens infecting S. paramamosain. PMID:26186350

  18. 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

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

    PubMed Central

    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

  20. Rafting rocks reveal marine biological dispersal: A case study using clasts from beach-cast macroalgal holdfasts

    NASA Astrophysics Data System (ADS)

    Garden, Christopher J.; Craw, Dave; Waters, Jonathan M.; Smith, Abigail

    2011-12-01

    Tracking and quantifying biological dispersal presents a major challenge in marine systems. Most existing methods for measuring dispersal are limited by poor resolution and/or high cost. Here we use geological data to quantify the frequency of long-distance dispersal in detached bull-kelp (Phaeophyceae: Durvillaea) in southern New Zealand. Geological resolution in this region is enhanced by the presence of a number of distinct and readily-identifiable geological terranes. We sampled 13,815 beach-cast bull-kelp plants across 130 km of coastline. Rocks were found attached to 2639 of the rafted plants, and were assigned to specific geological terranes (source regions) to quantify dispersal frequencies and distances. Although the majority of kelp-associated rock specimens were found to be locally-derived, a substantial number (4%) showed clear geological evidence of long-distance dispersal, several having travelled over 200 km from their original source regions. The proportion of local versus foreign clasts varied considerably between regions. While short-range dispersal clearly predominates, long-distance travel of detached bull-kelp plants is shown to be a common and ongoing process that has potential to connect isolated coastal populations. Geological analyses represent a cost-effective and powerful method for assigning large numbers of drifted macroalgae to their original source regions.

  1. Reactive oxygen species promote raft formation in T lymphocytes.

    PubMed

    Lu, Shu-Ping; Lin Feng, Ming-Hsien; Huang, Huey-Lan; Huang, Ya-Ching; Tsou, Wen-I; Lai, Ming-Zong

    2007-04-01

    Lipid rafts are involved in many cell biology events, yet the molecular mechanisms on how rafts are formed are poorly understood. In this study we probed the possible requirement of reactive oxygen species (ROS) for T-cell receptor (TCR)-induced lipid raft formation. Microscopy and biochemical analyses illustrated that blockage of ROS production, by superoxide dismutase-mimic MnTBAP, significantly reduced partitioning of LAT, phospho-LAT, and PLC-gamma in lipid rafts. Another antioxidant N-acetylcysteine (NAC) displayed a similar suppressive effect on the entry of phospho-LAT into raft microdomains. The involvement of ROS in TCR-mediated raft assembly was observed in T-cell hybridomas, T leukemia cells, and normal T cells. Removal of ROS was accompanied by an attenuated activation of LAT and PKCtheta, with reduced production of IL-2. Consistently, treating T cells with the ROS-producer tert-butyl hydrogen peroxide (TBHP) greatly enhanced membrane raft formation, distribution of phospho-LAT into lipid rafts, and increased IL-2 production. Our results indicate for the first time that ROS contribute to TCR-induced membrane raft formation. PMID:17349922

  2. Evidence that a lipolytic enzyme—hematopoietic-specific phospholipase C-β2—promotes mobilization of hematopoietic stem cells by decreasing their lipid raft-mediated bone marrow retention and increasing the promobilizing effects of granulocytes

    PubMed Central

    Adamiak, M; Poniewierska-Baran, A; Borkowska, S; Schneider, G; Abdelbaset-Ismail, A; Suszynska, M; Abdel-Latif, A; Kucia, M; Ratajczak, J; Ratajczak, M Z

    2016-01-01

    Hematopoietic stem/progenitor cells (HSPCs) reside in the bone marrow (BM) microenvironment and are retained there by the interaction of membrane lipid raft-associated receptors, such as the α-chemokine receptor CXCR4 and the α4β1-integrin (VLA-4, very late antigen 4 receptor) receptor, with their respective specific ligands, stromal-derived factor 1 and vascular cell adhesion molecule 1, expressed in BM stem cell niches. The integrity of the lipid rafts containing these receptors is maintained by the glycolipid glycosylphosphatidylinositol anchor (GPI-A). It has been reported that a cleavage fragment of the fifth component of the activated complement cascade, C5a, has an important role in mobilizing HSPCs into the peripheral blood (PB) by (i) inducing degranulation of BM-residing granulocytes and (ii) promoting their egress from the BM into the PB so that they permeabilize the endothelial barrier for subsequent egress of HSPCs. We report here that hematopoietic cell-specific phospholipase C-β2 (PLC-β2) has a crucial role in pharmacological mobilization of HSPCs. On the one hand, when released during degranulation of granulocytes, it digests GPI-A, thereby disrupting membrane lipid rafts and impairing retention of HSPCs in BM niches. On the other hand, it is an intracellular enzyme required for degranulation of granulocytes and their egress from BM. In support of this dual role, we demonstrate that PLC-β2-knockout mice are poor mobilizers and provide, for the first time, evidence for the involvement of this lipolytic enzyme in the mobilization of HSPCs. PMID:26582648

  3. Transforming Growth Factor-β1 (TGF-β1)-stimulated Fibroblast to Myofibroblast Differentiation Is Mediated by Hyaluronan (HA)-facilitated Epidermal Growth Factor Receptor (EGFR) and CD44 Co-localization in Lipid Rafts*

    PubMed Central

    Midgley, Adam C.; Rogers, Mathew; Hallett, Maurice B.; Clayton, Aled; Bowen, Timothy; Phillips, Aled O.; Steadman, Robert

    2013-01-01

    Fibroblast to myofibroblast differentiation drives effective wound healing and is largely regulated by the cytokine transforming growth factor-β1 (TGF-β1). Myofibroblasts express α-smooth muscle actin and are present in granulation tissue, where they are responsible for wound contraction. Our previous studies show that fibroblast differentiation in response to TGF-β1 is dependent on and mediated by the linear polysaccharide hyaluronan (HA). Both the HA receptor, CD44, and the epidermal growth factor receptor (EGFR) are involved in this differentiation response. The aim of this study was to understand the mechanisms linking HA-, CD44-, and EGFR-regulated TGF-β1-dependent differentiation. CD44 and EGFR co-localization within membrane-bound lipid rafts was necessary for differentiation, and this triggered downstream mitogen-activated protein kinase (MAPK/ERK) and Ca2+/calmodulin kinase II (CaMKII) activation. We also found that ERK phosphorylation was upstream of CaMKII phosphorylation, that ERK activation was necessary for CaMKII signaling, and that both kinases were essential for differentiation. In addition, HA synthase-2 (HAS2) siRNA attenuated both ERK and CaMKII signaling and sequestration of CD44 into lipid rafts, preventing differentiation. In summary, the data suggest that HAS2-dependent production of HA facilitates TGF-β1-dependent fibroblast differentiation through promoting CD44 interaction with EGFR held within membrane-bound lipid rafts. This induces MAPK/ERK, followed by CaMKII activation, leading to differentiation. This pathway is synergistic with the classical TGF-β1-dependent SMAD-signaling pathway and may provide a novel opportunity for intervention in wound healing. PMID:23589287

  4. Lipidome analysis reveals antifungal polyphenol curcumin affects membrane lipid homeostasis.

    PubMed

    Sharma, Monika; Dhamgaye, Sanjiveeni; Singh, Ashutosh; Prasad, Rajendra

    2012-01-01

    This study shows that antifungal curcumin (CUR), significantly depletes ergosterol levels in Candida albicans. CUR while displaying synergy with fluconazole (FLC) lowers ergosterol. However, CUR alone at its synergistic concentration (lower than MIC50), could not affect ergosterol contents. For deeper insight of CUR effects on lipids, we performed high throughput mass spectroscopy (MS) based lipid profiling of C. albicans cells. The lipidome analysis revealed that there were no major changes in phosphoglycerides (PGLs) composition following CUR treatment of Candida, however, significant differences in molecular species of PGLs were detected. Among major SPLs, CUR treatment resulted in the reduction of ceramide and accumulation of IPCs levels. The lipidome of CUR treated cells confirmed a dramatic drop in the ergosterol levels with a simultaneous accumulation of its biosynthetic precursors. This was further supported by the fact that the mutants defective in ergosterol biosynthesis (ERG2 and ERG11) and those lacking the transcription factor regulating ergosterol biosynthesis, UPC2, were highly susceptible to CUR. Our study first time shows that CUR, for its antifungal activity, targets and down regulates delta 5, 6 desaturase (ERG3) resulting in depletion of ergosterol. This results in parallel accumulation of ergosterol biosynthetic precursors, generation of reactive oxygen species (ROS) and cell death. PMID:22201946

  5. Fish oil increases raft size and membrane order of B cells accompanied by differential effects on function[S

    PubMed Central

    Rockett, Benjamin Drew; Teague, Heather; Harris, Mitchel; Melton, Mark; Williams, Justin; Wassall, Stephen R.; Shaikh, Saame Raza

    2012-01-01

    Fish oil (FO) targets lipid microdomain organization to suppress T-cell and macrophage function; however, little is known about this relationship with B cells, especially at the animal level. We previously established that a high FO dose diminished mouse B-cell lipid raft microdomain clustering induced by cross-linking GM1. To establish relevance, here we tested a FO dose modeling human intake on B-cell raft organization relative to a control. Biochemical analysis revealed more docosahexaenoic acid (DHA) incorporated into phosphatidylcholines than phosphatidylethanolamines of detergent-resistant membranes, consistent with supporting studies with model membranes. Subsequent imaging experiments demonstrated that FO increased raft size, GM1 expression, and membrane order upon cross-linking GM1 relative to no cross-linking. Comparative in vitro studies showed some biochemical differences from in vivo measurements but overall revealed that DHA, but not eicosapentaenoic acid (EPA), increased membrane order. Finally, we tested the hypothesis that disrupting rafts with FO would suppress B-cell responses ex vivo. FO enhanced LPS-induced B-cell activation but suppressed B-cell stimulation of transgenic naive CD4+ T cells. Altogether, our studies with B cells support an emerging model that FO increases raft size and membrane order accompanied by functional changes; furthermore, the results highlight differences in EPA and DHA bioactivity. PMID:22315394

  6. Solution Synchrotron X-ray Diffraction Reveals Structural Details of Lipid Domains in Ternary Mixtures

    SciTech Connect

    Yuan, J.; Kiss, A; Pramudya, Y; Nguyen, L; Hirst, L

    2009-01-01

    The influence of cholesterol on lipid bilayer structure is significant and the effect of cholesterol on lipid sorting and phase separation in lipid-raft-forming model membrane systems has been well investigated by microscopy methods on giant vesicles. An important consideration however is the influence of fluorescence illumination on the phase state of these lipids and this effect must be carefully minimized. In this paper, we show that synchrotron x-ray scattering on solution lipid mixtures is an effective alternative technique for the identification and characterization of the l o (liquid ordered) and l d (liquid disordered) phases. The high intensity of synchrotron x rays allows the observation of up to 5 orders of diffraction from the l o phase, whereas only two are clearly visible when the l d phase alone is present. This data can be collected in approximately 1 min/sample, allowing rapid generation of phase data. In this paper, we measure the lamellar spacing in both the liquid-ordered and liquid-disordered phases simultaneously, as a function of cholesterol concentration in two different ternary mixtures. We also observe evidence of a third gel-phaselike population at 10-12 mol % cholesterol and determine the thickness of the bilayer for this phase. Importantly we are able to look at phase coexistence in the membrane independent of photoeffects.

  7. Intracellular lipid flux and membrane microdomains as organizing principles in inflammatory cell signaling1

    PubMed Central

    Fessler, Michael B.; Parks, John S.

    2011-01-01

    Lipid rafts and caveolae play a pivotal role in organization of signaling by Toll-like Receptor (TLR)4 and several other immune receptors. Beyond the simple cataloguing of signaling events compartmentalized by these membrane microdomains, recent studies have revealed the surprisingly central importance of dynamic remodeling of membrane lipid domains to immune signaling. Simple interventions upon membrane lipid, such as changes in cholesterol loading or crosslinking of raft lipids, are sufficient to induce micron-scale reordering of membranes and their protein cargo with consequent signal transduction. In this review, using TLR signaling in the macrophage as a central focus, we discuss emerging evidence that environmental and genetic perturbations of membrane lipid regulate protein signaling, illustrate how homeostatic flow of cholesterol and other lipids through rafts regulates the innate immune response, and highlight recent attempts to harness these insights towards therapeutic development. PMID:21810617

  8. Raft-mediated Src homology 2 domain-containing proteintyrosine phosphatase 2 (SHP-2) regulation in microglia.

    PubMed

    Kim, Hee Young; Park, Soo Jung; Joe, Eun-hye; Jou, Ilo

    2006-04-28

    Janus kinase-signal transducer and activator of transcription (JAK-STAT) signals play important roles in cell proliferation, apoptosis, and inflammation, and they recently have been considered as therapeutic targets for suppressing oncogenesis and inflammatory process. Phosphatases including Src homology 2 domain-containing protein-tyrosine phosphatases (SHPs), are well known as negative regulators of the JAK-STAT pathway, but their precise mechanisms are largely unknown. Based on our previous finding that in cultured rat brain microglia, gangliosides induce rapid and transient activation of the JAK-STAT pathway, we hypothesized that raft-mediated SHP-2 activation is involved in transient activation of JAK-STAT signaling by gangliosides. We first used Western blot analysis to confirm that gangliosides rapidly induce the phosphorylation of SHP-2. This was inhibited by pretreatment with the lipid raft disrupter filipin and was restored following filipin removal. Immunostaining using antibodies directed against p-SHP-2 and flotillin-1 revealed ganglioside-induced clustering and polarization of p-SHP-2 in membrane rafts. Raft-associated regulation of SHP-2 was further demonstrated in fractionation experiments using detergent and detergent-free sucrose gradient ultracentrifugation. Rapid SHP-2 recruitment to detergent-insoluble raft fractions by gangliosides was inhibited by filipin, further indicating the involvement of rafts. We also confirmed by immunoprecipitation that SHP-2 rapidly binds in a raft-dependent manner to JAK2 in response to gangliosides. Our study therefore showed that transient activation of the JAK-STAT pathway by gangliosides is accomplished by SHP-2 in a raft-dependent manner in brain microglia. PMID:16507579

  9. Lipid-engineered Escherichia coli Membranes Reveal Critical Lipid Headgroup Size for Protein Function*

    PubMed Central

    Wikström, Malin; Kelly, Amélie A.; Georgiev, Alexander; Eriksson, Hanna M.; Klement, Maria Rosén; Bogdanov, Mikhail; Dowhan, William; Wieslander, Åke

    2009-01-01

    Escherichia coli membranes have a substantial bilayer curvature stress due to a large fraction of the nonbilayer-prone lipid phosphatidylethanolamine, and a mutant (AD93) lacking this lipid is severely crippled in several membrane-associated processes. Introduction of four lipid glycosyltransferases from Acholeplasma laidlawii and Arabidopsis thaliana, synthesizing large amounts of two nonbilayer-prone, and two bilayer-forming gluco- and galacto-lipids, (i) restored the curvature stress with the two nonbilayer lipids, and (ii) diluted the high negative lipid surface charge in all AD93 bilayers. Surprisingly, the bilayer-forming diglucosyl-diacylglycerol was almost as good in improving AD93 membrane processes as the two nonbilayer-prone glucosyl-diacylglycerol and galactosyl-diacylglycerol lipids, strongly suggesting that lipid surface charge dilution by these neutral lipids is very important for E. coli. Increased acyl chain length and unsaturation, plus cardiolipin (nonbilayer-prone) content, were probably also beneficial in the modified strains. However, despite a correct transmembrane topology for the transporter LacY in the diglucosyl-diacylglycerol clone, active transport failed in the absence of a nonbilayer-prone glycolipid. The corresponding digalactosyl-diacylglycerol bilayer lipid did not restore AD93 membrane processes, despite analogous acyl chain and cardiolipin contents. Chain ordering, probed by bis-pyrene lipids, was substantially lower in the digalactosyl-diacylglycerol strain lipids due to its extended headgroup. Hence, a low surface charge density of anionic lipids is important in E. coli membranes, but is inefficient if the headgroup of the diluting lipid is too large. This strongly indicates that a certain magnitude of the curvature stress is crucial for the bilayer in vivo. PMID:18981182

  10. Membrane rafts and caveolae in cardiovascular signaling

    PubMed Central

    Insel, Paul A.; Patel, Hemal H.

    2009-01-01

    Purpose of review Substantial evidence documents the key role of lipid (membrane) rafts and caveolae as microdomains that concentrate a wide variety of receptors and post-receptor components regulated by hormones, neurotransmitters and growth factors. Recent findings Recent data document that those microdomains are important in regulating vascular endothelial and smooth muscle cells and renal epithelial cells, and in particular in signal transduction across the plasma membrane. Summary Raft/caveolae domains are cellular regions, including in cardiovascular and renal epithelial cells, that organize a large number of signal transduction components, thereby providing spatially and temporally efficient regulation of cell function. PMID:19077689

  11. Lipidomics Analyses Reveal Temporal and Spatial Lipid Organization and Uncover Daily Oscillations in Intracellular Organelles.

    PubMed

    Aviram, Rona; Manella, Gal; Kopelman, Naama; Neufeld-Cohen, Adi; Zwighaft, Ziv; Elimelech, Meytar; Adamovich, Yaarit; Golik, Marina; Wang, Chunyan; Han, Xianlin; Asher, Gad

    2016-05-19

    Cells have evolved mechanisms to handle incompatible processes through temporal organization by circadian clocks and by spatial compartmentalization within organelles defined by lipid bilayers. Recent advances in lipidomics have led to identification of plentiful lipid species, yet our knowledge regarding their spatiotemporal organization is lagging behind. In this study, we quantitatively characterized the nuclear and mitochondrial lipidome in mouse liver throughout the day, upon different feeding regimens, and in clock-disrupted mice. Our analyses revealed potential connections between lipid species within and between lipid classes. Remarkably, we uncovered diurnal oscillations in lipid accumulation in the nucleus and mitochondria. These oscillations exhibited opposite phases and readily responded to feeding time. Furthermore, we found that the circadian clock coordinates the phase relation between the organelles. In summary, our study provides temporal and spatial depiction of lipid organization and reveals the presence and coordination of diurnal rhythmicity in intracellular organelles. PMID:27161994

  12. The ant raft

    NASA Astrophysics Data System (ADS)

    Mlot, Nathan; Hu, David; Equabai, Solomon

    2009-11-01

    To survive floods, fire ants link their arms together to assemble a raft with their own bodies. Because ants are nearly as dense as water, this cooperative behavior requires that a portion of the ant colony must sacrifice itself by remaining underwater to support the colony's weight. Surprisingly, few ants drown during this process due to a striking metamorphosis of the raft: as we show using time-lapse photography, the raft morphs from a spherical to a pancake shape. This pancake configuration--a monolayer of floating ants supporting their dry counterparts--allows all ants to both breathe and remain united as a colony. Data is presented in the form of the dimensions and the rates of formation of the ant raft. We use the statics of small floating bodies to account for the equilibrium raft size as a function of the initial mass and density of the ants.

  13. 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…

  14. 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.

  15. Nanoscale imaging reveals laterally expanding antimicrobial pores in lipid bilayers

    PubMed Central

    Rakowska, Paulina D.; Jiang, Haibo; Ray, Santanu; Pyne, Alice; Lamarre, Baptiste; Carr, Matthew; Judge, Peter J.; Ravi, Jascindra; M. Gerling, Ulla I.; Koksch, Beate; Martyna, Glenn J.; Hoogenboom, Bart W.; Watts, Anthony; Crain, Jason; Grovenor, Chris R. M.; Ryadnov, Maxim G.

    2013-01-01

    Antimicrobial peptides are postulated to disrupt microbial phospholipid membranes. The prevailing molecular model is based on the formation of stable or transient pores although the direct observation of the fundamental processes is lacking. By combining rational peptide design with topographical (atomic force microscopy) and chemical (nanoscale secondary ion mass spectrometry) imaging on the same samples, we show that pores formed by antimicrobial peptides in supported lipid bilayers are not necessarily limited to a particular diameter, nor they are transient, but can expand laterally at the nano-to-micrometer scale to the point of complete membrane disintegration. The results offer a mechanistic basis for membrane poration as a generic physicochemical process of cooperative and continuous peptide recruitment in the available phospholipid matrix. PMID:23671080

  16. Nanoscale imaging reveals laterally expanding antimicrobial pores in lipid bilayers.

    PubMed

    Rakowska, Paulina D; Jiang, Haibo; Ray, Santanu; Pyne, Alice; Lamarre, Baptiste; Carr, Matthew; Judge, Peter J; Ravi, Jascindra; Gerling, Ulla I M; Koksch, Beate; Martyna, Glenn J; Hoogenboom, Bart W; Watts, Anthony; Crain, Jason; Grovenor, Chris R M; Ryadnov, Maxim G

    2013-05-28

    Antimicrobial peptides are postulated to disrupt microbial phospholipid membranes. The prevailing molecular model is based on the formation of stable or transient pores although the direct observation of the fundamental processes is lacking. By combining rational peptide design with topographical (atomic force microscopy) and chemical (nanoscale secondary ion mass spectrometry) imaging on the same samples, we show that pores formed by antimicrobial peptides in supported lipid bilayers are not necessarily limited to a particular diameter, nor they are transient, but can expand laterally at the nano-to-micrometer scale to the point of complete membrane disintegration. The results offer a mechanistic basis for membrane poration as a generic physicochemical process of cooperative and continuous peptide recruitment in the available phospholipid matrix. PMID:23671080

  17. Comparative plant sphingolipidomic reveals specific lipids in seeds and oil.

    PubMed

    Tellier, Frédérique; Maia-Grondard, Alessandra; Schmitz-Afonso, Isabelle; Faure, Jean-Denis

    2014-07-01

    Plant sphingolipids are a highly diverse family of structural and signal lipids. Owing to their chemical diversity and complexity, a powerful analytical method was required to identify and quantify a large number of individual molecules with a high degree of structural accuracy. By using ultra-performance liquid chromatography with a single elution system coupled to electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) in the positive multiple reaction monitoring (MRM) mode, detailed sphingolipid composition was analyzed in various tissues of two Brassicaceae species Arabidopsis thaliana and Camelina sativa. A total of 300 molecular species were identified defining nine classes of sphingolipids, including Cers, hCers, Glcs and GIPCs. High-resolution mass spectrometry identified sphingolipids including amino- and N-acylated-GIPCs. The comparative analysis of seedling, seed and oil sphingolipids showed tissue specific distribution suggesting metabolic channeling and compartmentalization. PMID:24731258

  18. Mosquito, egg raft (image)

    MedlinePlus

    Mosquitoes of the Culex species lay their eggs in the form of egg rafts that float in ... feed on micro-organisms before developing into flying mosquitoes. (Image courtesy of the Centers for Disease Control ...

  19. Rafting in superalloys

    SciTech Connect

    Nabarro, F.R.N.

    1996-03-01

    The phenomenon of rafting in superalloys is described, with particular reference to modern superalloys with a high volume fraction of the particulate {gamma}{prime} phase. It is shown that in the elastic regime, the thermodynamic driving force for rafting is proportional to the applied stress, to the difference between the lattice parameters of the {gamma} matrix and the {gamma}{prime} particles, and to the difference of their elastic constants. A qualitative argument gives the sign of this driving force, which agrees with that determined by Pineau for a single isolated particle. Drawing on the work of Pollock and Argon and of Socrate and Parks, it is shown that after a plastic strain of the sample of order 2 {times} 10{sup {minus}4}, the driving force is proportional to the product of the applied stress and the lattice misfit, in agreement with the results of the calculations of Socrate and Parks. The rate of rafting is controlled by the diffusion of alloying elements. Here, the tendency of large atoms to move from regions of high hydrostatic pressure to those of low may outweigh the influence of concentration gradients. The deformation of the sample directly produced by rafting is small, of order 4.5 {times} 10{sup {minus}4}. The rafted structure is resistant to creep under low stresses at high temperatures. Under most experimental conditions at relatively high stresses, rafting accelerates creep; this effect may be less pronounced at the small strains acceptable under operational conditions.

  20. Lights on: Dye dequenching reveals polymersome fusion with polymer, lipid and stealth lipid vesicles

    DOE PAGESBeta

    Henderson, Ian M.; Collins, Aaron M.; Quintana, Hope A.; Montaño, Gabriel A.; Martinez, Julio A.; Paxton, Walter F.

    2015-12-13

    In this study, we develop a quantitative dye dequenching technique for the measurement of polymersome fusion, using it to characterize the salt mediated, mechanically-induced fusion of polymersomes with polymer, lipid, and so-called stealth lipid vesicles. While dye dequenching has been used to quantitatively explore liposome fusion in the past, this is the first use of dye dequenching to measure polymersome fusion of which we are aware. In addition to providing quantitative results, dye dequenching is ideal for detecting fusion in instances where DLS results would be ambiguous, such as low yield levels and size ranges outside the capabilities of DLS.more » The dye chosen for this study was a cyanine derivative, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR), which proved to provide excellent data on the extent of polymersome fusion. Using this technique, we have shown the limited fusion capabilities of polymersome/liposome heterofusion, notably DOPC vesicles fusing with polymersomes at half the efficiency of polymersome homofusion and DPPC vesicles showing virtually no fusion. In addition to these key heterofusion experiments, we determined the broad applicability of dye dequenching in measuring kinetic rates of polymersome fusion; and showed that even at elevated temperatures or over multiple weeks' time, no polymersome fusion occurred without agitation. Stealth liposomes formed from DPPC and PEO-functionalized lipid, however, fused with polymersomes and stealth liposomes with relatively high efficiency, lending support to our hypothesis that the response of the PEO corona to salt is a key factor in the fusion process. This last finding suggests that although the conjugation of PEO to lipids increases vesicle biocompatibility and enables their longer circulation times, it also renders the vesicles subject to destabilization under high salt and shear (e.g. in the circulatory system) that may lead to, in this case, fusion.« less

  1. Lights on: Dye dequenching reveals polymersome fusion with polymer, lipid and stealth lipid vesicles

    SciTech Connect

    Henderson, Ian M.; Collins, Aaron M.; Quintana, Hope A.; Montaño, Gabriel A.; Martinez, Julio A.; Paxton, Walter F.

    2015-12-13

    In this study, we develop a quantitative dye dequenching technique for the measurement of polymersome fusion, using it to characterize the salt mediated, mechanically-induced fusion of polymersomes with polymer, lipid, and so-called stealth lipid vesicles. While dye dequenching has been used to quantitatively explore liposome fusion in the past, this is the first use of dye dequenching to measure polymersome fusion of which we are aware. In addition to providing quantitative results, dye dequenching is ideal for detecting fusion in instances where DLS results would be ambiguous, such as low yield levels and size ranges outside the capabilities of DLS. The dye chosen for this study was a cyanine derivative, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR), which proved to provide excellent data on the extent of polymersome fusion. Using this technique, we have shown the limited fusion capabilities of polymersome/liposome heterofusion, notably DOPC vesicles fusing with polymersomes at half the efficiency of polymersome homofusion and DPPC vesicles showing virtually no fusion. In addition to these key heterofusion experiments, we determined the broad applicability of dye dequenching in measuring kinetic rates of polymersome fusion; and showed that even at elevated temperatures or over multiple weeks' time, no polymersome fusion occurred without agitation. Stealth liposomes formed from DPPC and PEO-functionalized lipid, however, fused with polymersomes and stealth liposomes with relatively high efficiency, lending support to our hypothesis that the response of the PEO corona to salt is a key factor in the fusion process. This last finding suggests that although the conjugation of PEO to lipids increases vesicle biocompatibility and enables their longer circulation times, it also renders the vesicles subject to destabilization under high salt and shear (e.g. in the circulatory system) that may lead to, in this case, fusion.

  2. Cholesterol overload induces apoptosis in SH-SY5Y human neuroblastoma cells through the up regulation of flotillin-2 in the lipid raft and the activation of BDNF/Trkb signaling.

    PubMed

    Huang, Yen-Ning; Lin, Ching-I; Liao, Hsiang; Liu, Chin-Yu; Chen, Yue-Hua; Chiu, Wan-Chun; Lin, Shyh-Hsiang

    2016-07-22

    Epidemiological investigations have shown that Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. It has been indicated that the cholesterol concentration in the brain of AD patients is higher than that in normal people. In this study, we investigated the effects of cholesterol concentrations, 0, as the control, 3.125, 12.5, and 25μM, on cholesterol metabolism, neuron survival, AD-related protein expressions, and cell morphology and apoptosis using SH-SY5Y human neuroblastoma cells. We observed that expressions of cholesterol hydroxylase (Cyp46), flotillin-2 (a marker of lipid raft content), and truncated tyrosine kinase B (TrkBtc) increased, while expressions of brain-derived neurotrophic factor (BDNF) and full-length TrkB (TrkBfl) decreased as the concentration of cholesterol loading increased. Down-regulation of the PI3K-Akt-glycogen synthase kinase (GSK)-3β cascade and cell apoptosis were also observed at higher concentrations of cholesterol, along with elevated levels of β-amyloid (Aβ), β-secretase (BACE), and reactive oxygen species (ROS). In conclusion, we found that cholesterol overload in neuronal cells imbalanced the cholesterol homeostasis and increased the protein expressions causing cell apoptosis, which illustrates the neurodegenerative pathology of abnormally elevated cholesterol concentrations found in AD patients. PMID:27155148

  3. Metabolic phenotyping reveals a lipid mediator response to ionizing radiation.

    PubMed

    Laiakis, Evagelia C; Strassburg, Katrin; Bogumil, Ralf; Lai, Steven; Vreeken, Rob J; Hankemeier, Thomas; Langridge, James; Plumb, Robert S; Fornace, Albert J; Astarita, Giuseppe

    2014-09-01

    Exposure to ionizing radiation has dramatically increased in modern society, raising serious health concerns. The molecular response to ionizing radiation, however, is still not completely understood. Here, we screened mouse serum for metabolic alterations following an acute exposure to γ radiation using a multiplatform mass-spectrometry-based strategy. A global, molecular profiling revealed that mouse serum undergoes a series of significant molecular alterations following radiation exposure. We identified and quantified bioactive metabolites belonging to key biochemical pathways and low-abundance, oxygenated, polyunsaturated fatty acids (PUFAs) in the two groups of animals. Exposure to γ radiation induced a significant increase in the serum levels of ether phosphatidylcholines (PCs) while decreasing the levels of diacyl PCs carrying PUFAs. In exposed mice, levels of pro-inflammatory, oxygenated metabolites of arachidonic acid increased, whereas levels of anti-inflammatory metabolites of omega-3 PUFAs decreased. Our results indicate a specific serum lipidomic biosignature that could be utilized as an indicator of radiation exposure and as novel target for therapeutic intervention. Monitoring such a molecular response to radiation exposure might have implications not only for radiation pathology but also for countermeasures and personalized medicine. PMID:25126707

  4. NMR spectroscopy reveals the presence and association of lipids and keratin in adhesive gecko setae.

    PubMed

    Jain, Dharamdeep; Stark, Alyssa Y; Niewiarowski, Peter H; Miyoshi, Toshikazu; Dhinojwala, Ali

    2015-01-01

    Lipid and protein aggregates are one of the fundamental materials of biological systems. Examples include cell membranes, insect cuticle, vertebrate epidermis, feathers, hair and adhesive structures known as 'setae' on gecko toes. Until recently gecko setae were assumed to be composed entirely of keratin, but analysis of footprints left behind by geckos walking on surfaces revealed that setae include various kinds of lipids. However, the arrangement and molecular-level behavior of lipids and keratin in the setae is still not known. In the present study we demonstrate, for the first time, the use of Nuclear Magnetic Resonance (NMR) spectroscopy techniques to confirm the presence of lipids and investigate their association with keratin in 'pristine' sheds, or natural molts of the adhesive toe pad and non-adhesive regions of the skin. Analysis was also carried on the sheds after they were 'delipidized' to remove surface lipids. Our results show a distribution of similar lipids in both the skin and toe shed but with different dynamics at a molecular level. The present study can help us understand the gecko system both biologically and for design of synthetic adhesives, but the findings may be relevant to the characteristics of lipid-protein interactions in other biological systems. PMID:25902194

  5. Energetic modeling and single-molecule verification of dynamic regulation on receptor protein diffusion by actin corrals and lipid raft domains receptor

    NASA Astrophysics Data System (ADS)

    Lin, Chien Yu; Huang, Jung Y.; Lo, Leu-Wei

    2015-03-01

    To faithfully estimate a signal that varies in both space and time, the optimization strategy used by a live cell is to organize a collection of distributed and mobile receptors into a mobile active clustering. However, living eukaryotic cells are highly heterogeneous and stochastically dynamic. It is therefore important to develop an energetic model based on fundamental laws to verify that the underlying processes are energetically favorable. We developed an energetic model based on the generalized Langevin equation and the Cahn-Hilliard equation to simulate the diffusive behaviors of receptor proteins in the plasma membrane with a hierarchical structure of actin corrals, lipid domains, and receptor proteins. Single-molecule tracking data of EGFR acquired on live HeLa cells agrees with the simulation results. We discovered that after ligand binding, EGFR molecules move into lipid nanodomains. The transition rates between different diffusion states of liganded EGFR molecules are regulated by the lipid domains. Our method captures both the sensitivity of single-molecule processes, statistic accuracy of data analysis, and the hierarchical structure of plasma membranes.

  6. Disrupting membrane raft domains by alkylphospholipids.

    PubMed

    Gomide, A B; Thomé, C H; dos Santos, G A; Ferreira, G A; Faça, V M; Rego, E M; Greene, L J; Stabeli, R G; Ciancaglini, P; Itri, R

    2013-05-01

    Using phase contrast and fluorescence microscopy we study the influence of the alkylphospholipid, ALP, 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate, ODPC, in giant unilamellar vesicles, GUVs, composed of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), brain sphingomyelin (SM) and cholesterol (Chol). The results show that adding 100μM ODPC (below CMC) to the outer solution of GUVs promotes DOPC membrane disruption over a period of 1h of continuous observation. On the other hand, the presence of SM and Chol in homogeneous fluid lipid bilayers protects the membrane from disruption. Interestingly, by adding 100μM ODPC to GUVs containing DOPC:SM:Chol (1:1:1), which display liquid ordered (Lo)-liquid disordered (Ld) phase coexistence, the domains rapidly disappear in less than 1min of ODPC contact with the membrane. The lipids are subsequently redistributed to liquid domains within a time course of 14-18min, reflecting that the homogenous phase was not thermodynamically stable, followed by rupture of the GUVs. A similar mechanism of action is also observed for perifosine, although to a larger extent. Therefore, the initial stage of lipid raft disruption by both ODPC and perifosine, and maybe other ALPS, by promoting lipid mixing, may be correlated with their toxicity upon neoplastic cells, since selective (dis)association of essential proteins within lipid raft microdomains must take place in the plasma membrane. PMID:23376656

  7. Anchorage of HIV on permissive cells leads to coaggregation of viral particles with surface nucleolin at membrane raft microdomains.

    PubMed

    Nisole, Sébastien; Krust, Bernard; Hovanessian, Ara G

    2002-06-10

    The cross-linking of HIV on permissive cells results aggregation of HIV particles with surface nucleolin, CD4, and CXCR4, but without affecting the organization of CD45. In addition, HIV particles and nucleolin coaggregate with glycolipid-enriched membrane microdomains (GEMs) containing ganglioside, and glycosylphosphatidylinositol-linked proteins CD90 and CD59, pointing out that HIV anchorage induces lateral assemblies of specific membrane components into lipid rafts in which surface nucleolin is also incorporated. Consequently, equilibrium density fractionation of extracts from infected cells revealed that HIV proteins and nucleolin copurify with Triton X-100-resistant GEM-associated proteins. After HIV entry, nucleolin is recovered also in fractions containing HIV DNA, viral matrix, and reverse transcriptase, thus suggesting that it could accompany viral entry. We show that surface nucleolin is markedly down-regulated a few hours following HIV entry into permissive cells; an effect that appears to be the consequence of its translocation into the cytoplasm. Our findings demonstrate that anchorage of HIV particles on permissive cells induces aggegation of surface nucleolin and its association with detergent-insoluble lipid raft components. Moreover, they support the suggestion that surface nucleolin and lipid rafts are implicated in early events in the HIV entry process. PMID:12027446

  8. 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

  9. STED Nanoscopy Reveals Molecular Details of Cholesterol- and Cytoskeleton-Modulated Lipid Interactions in Living Cells

    PubMed Central

    Mueller, V.; Ringemann, C.; Honigmann, A.; Schwarzmann, G.; Medda, R.; Leutenegger, M.; Polyakova, S.; Belov, V.N.; Hell, S.W.; Eggeling, C.

    2011-01-01

    Details about molecular membrane dynamics in living cells, such as lipid-protein interactions, are often hidden from the observer because of the limited spatial resolution of conventional far-field optical microscopy. The superior spatial resolution of stimulated emission depletion (STED) nanoscopy can provide new insights into this process. The application of fluorescence correlation spectroscopy (FCS) in focal spots continuously tuned down to 30 nm in diameter distinguishes between free and anomalous molecular diffusion due to, for example, transient binding of lipids to other membrane constituents, such as lipids and proteins. We compared STED-FCS data recorded on various fluorescent lipid analogs in the plasma membrane of living mammalian cells. Our results demonstrate details about the observed transient formation of molecular complexes. The diffusion characteristics of phosphoglycerolipids without hydroxyl-containing headgroups revealed weak interactions. The strongest interactions were observed with sphingolipid analogs, which showed cholesterol-assisted and cytoskeleton-dependent binding. The hydroxyl-containing headgroup of gangliosides, galactosylceramide, and phosphoinositol assisted binding, but in a much less cholesterol- and cytoskeleton-dependent manner. The observed anomalous diffusion indicates lipid-specific transient hydrogen bonding to other membrane molecules, such as proteins, and points to a distinct connectivity of the various lipids to other membrane constituents. This strong interaction is different from that responsible for forming cholesterol-dependent, liquid-ordered domains in model membranes. PMID:21961591

  10. 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.

  11. 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

  12. The Lipid-Droplet Proteome Reveals that Droplets Are a Protein-Storage Depot

    SciTech Connect

    Cermelli, Silvia; Guo, Yi; Gross, Steven P.; Welte, Michael

    2006-09-19

    Lipid droplets are ubiquitous organelles that are among the basic building blocks of eukaryotic cells. Despite central roles for cholesterol homeostasis and lipid metabolism, their function and protein composition are poorly understood. Results: We purified lipid droplets from Drosophila embryos and analyzed the associated proteins by capillary LC-MS-MS. Important functional groups include enzymes involved in lipid metabolism, signaling molecules, and proteins related to membrane trafficking. Unexpectedly, histones H2A, H2Av, and H2B were present. Using biochemistry, genetics, real-time imaging, and cell biology, we confirm that roughly 50% of certain embryonic histones are physically attached to lipid droplets, a localization conserved in other fly species. Histone association with droplets starts during oogenesis and is prominent in early embryos, but it is undetectable in later stages or in cultured cells. Histones on droplets are not irreversibly trapped; quantitation of droplet histone levels and transplantation experiments suggest that histones are transferred from droplets to nuclei as development proceeds. When this maternal store of histones is unavailable because lipid droplets are mislocalized, zygotic histone production starts prematurely. Conclusions: Because we uncover a striking proteomic similarity of Drosophila droplets to mammalian lipid droplets, Drosophila likely provides a good model for understanding droplet function in general. Our analysis also reveals a new function for these organelles; the massive nature of histone association with droplets and its developmental time-course suggest that droplets sequester maternally provided proteins until they are needed. We propose that lipid droplets can serve as transient storage depots for proteins that lack appropriate binding partners in the cell. Such sequestration may provide a general cellular strategy for handling excess proteins.

  13. The Basic Domain of HIV-Tat Transactivating Protein Is Essential for Its Targeting to Lipid Rafts and Regulating Fibroblast Growth Factor-2 Signaling in Podocytes Isolated from Children with HIV-1–Associated Nephropathy

    PubMed Central

    Xie, Xuefang; Colberg-Poley, Anamaris M.; Das, Jharna R.; Li, Jinliang; Zhang, Aiping; Tang, Pingtao; Jerebtsova, Marina; Gutkind, J. Silvio

    2014-01-01

    Podocyte injury has a critical role in the pathogenesis of HIV-associated nephropathy (HIVAN). The HIV-1 transactivator of transcription (Tat), combined with fibroblast growth factor-2 (FGF-2), can induce the dedifferentiation and proliferation of cultured human podocytes. Cellular internalization of Tat requires interactions with heparan sulfate proteoglycans and cholesterol-enriched lipid rafts (LRs). However, the specific distribution of Tat in human podocytes and its ability to associate with LRs have not been documented. Here, we found that Tat is preferentially recruited to LRs in podocytes isolated from children with HIVAN. Furthermore, we identified arginines in the basic domain (RKKRRQRRR) of Tat as essential for (1) targeting Tat to LRs, (2) Tat-mediated increases in the expression of Rho-A and matrix metalloproteinase-9 in LRs, and (3) Tat-mediated enhancement of FGF-2 signaling in human podocytes and HIV-transgenic mouse kidneys and the exacerbation of renal lesions in these mice. Tat carrying alanine substitutions in the basic domain (AKKAAQAAA) remained localized in the cytosol and did not associate with LRs or enhance FGF-2 signaling in cultured podocytes. These results show the specific association of Tat with LRs in podocytes isolated from children with HIVAN, confirm Tat as a regulator of FGF-2 signaling in LRs, and identify the key domain of Tat responsible for promoting these effects and aggravating renal injury in HIV-transgenic mice. Moreover, these results provide a molecular framework for developing novel therapies to improve the clinical outcome of children with HIVAN. PMID:24578133

  14. Sphingolipid–Cholesterol Rafts Diffuse as Small Entities in the Plasma Membrane of Mammalian Cells

    PubMed Central

    Pralle, A.; Keller, P.; Florin, E.-L.; Simons, K.; Hörber, J.K.H.

    2000-01-01

    To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam ≤ 100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 ± 13 nm in size diffusing as one entity for minutes. PMID:10704449

  15. Exome-wide association analysis reveals novel coding sequence variants associated with lipid traits in Chinese.

    PubMed

    Tang, Clara S; Zhang, He; Cheung, Chloe Y Y; Xu, Ming; Ho, Jenny C Y; Zhou, Wei; Cherny, Stacey S; Zhang, Yan; Holmen, Oddgeir; Au, Ka-Wing; Yu, Haiyi; Xu, Lin; Jia, Jia; Porsch, Robert M; Sun, Lijie; Xu, Weixian; Zheng, Huiping; Wong, Lai-Yung; Mu, Yiming; Dou, Jingtao; Fong, Carol H Y; Wang, Shuyu; Hong, Xueyu; Dong, Liguang; Liao, Yanhua; Wang, Jiansong; Lam, Levina S M; Su, Xi; Yan, Hua; Yang, Min-Lee; Chen, Jin; Siu, Chung-Wah; Xie, Gaoqiang; Woo, Yu-Cho; Wu, Yangfeng; Tan, Kathryn C B; Hveem, Kristian; Cheung, Bernard M Y; Zöllner, Sebastian; Xu, Aimin; Eugene Chen, Y; Jiang, Chao Qiang; Zhang, Youyi; Lam, Tai-Hing; Ganesh, Santhi K; Huo, Yong; Sham, Pak C; Lam, Karen S L; Willer, Cristen J; Tse, Hung-Fat; Gao, Wei

    2015-01-01

    Blood lipids are important risk factors for coronary artery disease (CAD). Here we perform an exome-wide association study by genotyping 12,685 Chinese, using a custom Illumina HumanExome BeadChip, to identify additional loci influencing lipid levels. Single-variant association analysis on 65,671 single nucleotide polymorphisms reveals 19 loci associated with lipids at exome-wide significance (P<2.69 × 10(-7)), including three Asian-specific coding variants in known genes (CETP p.Asp459Gly, PCSK9 p.Arg93Cys and LDLR p.Arg257Trp). Furthermore, missense variants at two novel loci-PNPLA3 p.Ile148Met and PKD1L3 p.Thr429Ser-also influence levels of triglycerides and low-density lipoprotein cholesterol, respectively. Another novel gene, TEAD2, is found to be associated with high-density lipoprotein cholesterol through gene-based association analysis. Most of these newly identified coding variants show suggestive association (P<0.05) with CAD. These findings demonstrate that exome-wide genotyping on samples of non-European ancestry can identify additional population-specific possible causal variants, shedding light on novel lipid biology and CAD. PMID:26690388

  16. Exome-wide association analysis reveals novel coding sequence variants associated with lipid traits in Chinese

    PubMed Central

    Tang, Clara S.; Zhang, He; Cheung, Chloe Y. Y.; Xu, Ming; Ho, Jenny C. Y.; Zhou, Wei; Cherny, Stacey S.; Zhang, Yan; Holmen, Oddgeir; Au, Ka-Wing; Yu, Haiyi; Xu, Lin; Jia, Jia; Porsch, Robert M.; Sun, Lijie; Xu, Weixian; Zheng, Huiping; Wong, Lai-Yung; Mu, Yiming; Dou, Jingtao; Fong, Carol H. Y.; Wang, Shuyu; Hong, Xueyu; Dong, Liguang; Liao, Yanhua; Wang, Jiansong; Lam, Levina S. M.; Su, Xi; Yan, Hua; Yang, Min-Lee; Chen, Jin; Siu, Chung-Wah; Xie, Gaoqiang; Woo, Yu-Cho; Wu, Yangfeng; Tan, Kathryn C. B.; Hveem, Kristian; Cheung, Bernard M. Y.; Zöllner, Sebastian; Xu, Aimin; Eugene Chen, Y; Jiang, Chao Qiang; Zhang, Youyi; Lam, Tai-Hing; Ganesh, Santhi K.; Huo, Yong; Sham, Pak C.; Lam, Karen S. L.; Willer, Cristen J.; Tse, Hung-Fat; Gao, Wei

    2015-01-01

    Blood lipids are important risk factors for coronary artery disease (CAD). Here we perform an exome-wide association study by genotyping 12,685 Chinese, using a custom Illumina HumanExome BeadChip, to identify additional loci influencing lipid levels. Single-variant association analysis on 65,671 single nucleotide polymorphisms reveals 19 loci associated with lipids at exome-wide significance (P<2.69 × 10−7), including three Asian-specific coding variants in known genes (CETP p.Asp459Gly, PCSK9 p.Arg93Cys and LDLR p.Arg257Trp). Furthermore, missense variants at two novel loci—PNPLA3 p.Ile148Met and PKD1L3 p.Thr429Ser—also influence levels of triglycerides and low-density lipoprotein cholesterol, respectively. Another novel gene, TEAD2, is found to be associated with high-density lipoprotein cholesterol through gene-based association analysis. Most of these newly identified coding variants show suggestive association (P<0.05) with CAD. These findings demonstrate that exome-wide genotyping on samples of non-European ancestry can identify additional population-specific possible causal variants, shedding light on novel lipid biology and CAD. PMID:26690388

  17. Structure and dynamics of nano-sized raft-like domains on the plasma membrane

    NASA Astrophysics Data System (ADS)

    Herrera, Fernando E.; Pantano, Sergio

    2012-01-01

    Cell membranes are constitutively composed of thousands of different lipidic species, whose specific organization leads to functional heterogeneities. In particular, sphingolipids, cholesterol and some proteins associate among them to form stable nanoscale domains involved in recognition, signaling, membrane trafficking, etc. Atomic-detail information in the nanometer/second scale is still elusive to experimental techniques. In this context, molecular simulations on membrane systems have provided useful insights contributing to bridge this gap. Here we present the results of a series of simulations of biomembranes representing non-raft and raft-like nano-sized domains in order to analyze the particular structural and dynamical properties of these domains. Our results indicate that the smallest (5 nm) raft domains are able to preserve their distinctive structural and dynamical features, such as an increased thickness, higher ordering, lower lateral diffusion, and specific lipid-ion interactions. The insertion of a transmembrane protein helix into non-raft, extended raft-like, and raft-like nanodomain environments result in markedly different protein orientations, highlighting the interplay between the lipid-lipid and lipid-protein interactions.

  18. Targeting the Raft-Associated Akt Signaling in Hepatocellular Carcinoma

    PubMed Central

    Liu, Yuan; Lv, Ji-Yun; Shi, Jian-Fei; Yang, Mei; Liu, Shu-Hong; Li, Zhi-Wei; Wang, Hong-Bo; Zhang, Shao-Geng; Liu, Zhen-Wen; Ding, Jin-Biao; Xu, Dong-Ping; Zhao, Jing-Min

    2014-01-01

    Caveolin-1 and flotillin-1 are considered as markers of lipid rafts which can be regarded as sorting platforms for targeted transport of transmembrane proteins and are involved in fundamental cellular events such as signal transduction, cell adhesion, lipid/protein sorting, and human cancer. We addressed caveolin-1 and flotillin-1 expression in 90 human hepatocellular carcinoma (HCC) and adjacent noncancerous tissues (ANT) samples by SDS-PAGE and immunoblotting with specific antibodies. Significant caveolin-1 and flotillin-1 overexpression was found in HCC tissues compared to ANT and was confirmed by immunohistochemistry. Raft-associated Akt signaling pathway components involved in the regulation of cell survival were altered by western blotting in HCC microdomain-enriched subcellular fractions purified from paired HCC and ANT samples. Our results demonstrated that the activity of raft-associated but not total membrane Akt determines its cellular functions. Lipid rafts differ in different types of tissues, which allows for the possibility of tissue-type-specific targeting for cell survival. PMID:25243186

  19. Plasma membrane rafts of rainbow trout are subject to thermal acclimation.

    PubMed

    Zehmer, John K; Hazel, Jeffrey R

    2003-05-01

    Rafts are cholesterol- and sphingolipid-enriched microdomains of the plasma membrane (PM) that organize many signal transduction pathways. Interactions between cholesterol and saturated lipids lead to patches of liquid-ordered membrane (rafts) phase-separating from the remaining PM. Phase behavior is temperature sensitive, and acute changes in temperature experienced by poikilotherms would be expected to perturb raft structure, necessitating an acclimatory response. Therefore, with thermal acclimation, we would expect compositional changes in the raft directed to offset this perturbation. Using differential and density gradient centrifugation, we separated PM from the livers of rainbow trout acclimated to 5 degrees C and 20 degrees C into raft-enriched (raft) and raft-depleted PM (RDPM). Compared with RDPM, the raft fractions were enriched in cholesterol, the beta(2)-adrenergic receptor and adenylyl cyclase, which are commonly used markers for this microdomain. Furthermore, cholesterol was enriched in all fractions from warm-compared with cold-acclimated animals, but this increase was 3.4 times greater in raft than in PM. We developed a novel approach for measuring membrane molecular interaction strength (and thus the tendency to stabilize raft structure) based on the susceptibility of membranes to detergent. Specifically, studies with model vesicles demonstrated that the capacity of a membrane to accommodate detergent prior to solubilization (saturation point) was a good index of this property. The saturation point of the isolated membrane preparations was temperature sensitive and was significantly different in 5 degrees C- and 20 degrees C-acclimated RDPM when assayed at 5 degrees C and 20 degrees C, respectively. By contrast, this comparison in rafts was not significantly different, suggesting compensation of this property. These data suggest that compositional changes made in the PM during thermal acclimation act to offset thermal perturbation of the raft but

  20. Transcriptome Analysis Reveals Regulation of Gene Expression for Lipid Catabolism in Young Broilers by Butyrate Glycerides

    PubMed Central

    Yin, Fugui; Yu, Hai; Lepp, Dion; Shi, Xuejiang; Yang, Xiaojian; Hu, Jielun; Leeson, Steve; Yang, Chengbo; Nie, Shaoping; Hou, Yongqing; Gong, Joshua

    2016-01-01

    indicated that dietary BG intervention induced 79 and 205 characterized DEGs in the jejunum and liver, respectively. In addition, 255 and 165 TSEGs were detected in the liver and jejunum of BG-fed group, while 162 and 211 TSEGs genes were observed in the liver and jejunum of BD-fed birds, respectively. Bioinformatic analysis with both IPA and DAVID-BR further revealed a significant enrichment of DEGs and TSEGs in the biological processes for reducing the synthesis, storage, transportation and secretion of lipids in the jejunum, while those in the liver were for enhancing the oxidation of ingested lipids and fatty acids. In particular, transcriptional regulators of THRSP and EGR-1 as well as several DEGs involved in the PPAR-α signaling pathway were significantly induced by dietary BG intervention for lipid catabolism. Conclusions Our results demonstrate that BG reduces body fat deposition via regulation of gene expression, which is involved in the biological events relating to the reduction of synthesis, storage, transportation and secretion, and improvement of oxidation of lipids and fatty acids. PMID:27508934

  1. Genetic Analysis of Arabidopsis Mutants Impaired in Plastid Lipid Import Reveals a Role of Membrane Lipids in Chloroplast Division

    SciTech Connect

    Fan, J.; Xu, C.

    2011-03-01

    The biogenesis of photosynthetic membranes in plants relies largely on lipid import from the endoplasmic reticulum (ER) and this lipid transport process is mediated by TGD proteins in Arabidopsis. Such a dependency of chloroplast biogenesis on ER-to-plastid lipid transport was recently exemplified by analyzing double mutants between tgd1-1 or tgd4-3 and fad6 mutants. The fad6 mutants are defective in the desaturation of membrane lipids in chloroplasts and therefore dependent on import of polyunsaturated lipid precursors from the ER for constructing a competent thylakoid membrane system. In support of a critical role of TGD proteins in ER-to-plastid lipid trafficking, we showed that the introduction of the tgd mutations into fad6 mutant backgrounds led to drastic reductions in relative amounts of thylakoid lipids. Moreover, the tgd1-1 fad6 and tgd4-3 fad6 double mutants were deficient in polyunsaturated fatty acids in chloroplast membrane lipids, and severely compromised in the biogenesis of photosynthetic membrane systems. Here we report that these double mutants are severely impaired in chloroplast division. The possible role of membrane lipids in chloroplast division is discussed.

  2. Association of Influenza Virus Proteins with Membrane Rafts

    PubMed Central

    Veit, Michael; Thaa, Bastian

    2011-01-01

    Assembly and budding of influenza virus proceeds in the viral budozone, a domain in the plasma membrane with characteristics of cholesterol/sphingolipid-rich membrane rafts. The viral transmembrane glycoproteins hemagglutinin (HA) and neuraminidase (NA) are intrinsically targeted to these domains, while M2 is seemingly targeted to the edge of the budozone. Virus assembly is orchestrated by the matrix protein M1, binding to all viral components and the membrane. Budding progresses by protein- and lipid-mediated membrane bending and particle scission probably mediated by M2. Here, we summarize the experimental evidence for this model with emphasis on the raft-targeting features of HA, NA, and M2 and review the functional importance of raft domains for viral protein transport, assembly and budding, environmental stability, and membrane fusion. PMID:22312341

  3. Differential proteomic analysis of STAT6 knockout mice reveals new regulatory function in liver lipid homeostasis.

    PubMed

    Iff, Joël; Wang, Wei; Sajic, Tatjana; Oudry, Nathalie; Gueneau, Estelle; Hopfgartner, Gérard; Varesio, Emmanuel; Szanto, Ildiko

    2009-10-01

    Increased inflammatory signaling is a key feature of metabolic disorders. In this context, the role of increased pro-inflammatory signals has been extensively studied. By contrast, no efforts have been dedicated to study the contrasting scenario: the attenuation of anti-inflammatory signals and their role in metabolic homeostasis. IL-4 and IL-13 are anti-inflammatory cytokines signaling through the Signal Transducer and Activator of Transcription 6 (STAT6). Our study was aimed at evaluating the lack of STAT6 signaling on liver homeostasis. To this end we analyzed the liver proteome of wild type and STAT6 knock-out mice using 2D nanoscale LC-MS/MS with iTRAQ labeling technique. The coordinated changes in proteins identified by this quantitative proteome analysis indicated disturbed lipid homeostasis and a state of hepatocellular stress. Most significantly, the expression of the liver fatty acid binding protein (FABP1) was increased in the knock-out mice. In line with the elevated FABP1 expression we found latent liver lipid accumulation in the STAT6-deficient mice which was further aggravated when mice were challenged by a high fat diet. In conclusion, our study revealed a so far uncharacterized role for STAT6 in regulating liver lipid homeostasis and demonstrates the importance of anti-inflammatory signaling in the defense against the development of liver steatosis. PMID:19663508

  4. 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.

  5. Phloem proteomics reveals new lipid-binding proteins with a putative role in lipid-mediated signaling

    DOE PAGESBeta

    Barbaglia, Allison M.; Tamot, Banita; Greve, Veronica; Hoffmann-Benning, Susanne

    2016-04-28

    Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and thatmore » they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all

  6. Phloem Proteomics Reveals New Lipid-Binding Proteins with a Putative Role in Lipid-Mediated Signaling.

    PubMed

    Barbaglia, Allison M; Tamot, Banita; Greve, Veronica; Hoffmann-Benning, Susanne

    2016-01-01

    Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and that they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all three

  7. Phloem Proteomics Reveals New Lipid-Binding Proteins with a Putative Role in Lipid-Mediated Signaling

    PubMed Central

    Barbaglia, Allison M.; Tamot, Banita; Greve, Veronica; Hoffmann-Benning, Susanne

    2016-01-01

    Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and that they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all three

  8. Capillary rafts and their destabilization

    NASA Astrophysics Data System (ADS)

    Protiere, Suzie; Abkarian, Manouk; Aristoff, Jeffrey; Stone, Howard

    2010-11-01

    Small objects trapped at an interface are very common in Nature (insects walking on water, ant rafts, bubbles or pollen at the water-air interface, membranes...) and are found in many multiphase industrial processes. The study of such particle-laden interfaces is therefore of practical as well as fundamental importance. Here we report experiments on the self-assembly of spherical particles into capillary rafts at an oil-water interface and elucidate how such rafts sink. We characterize different types of sinking behavior and show that it is possible to obtain "armored droplets," whereby the sinking oil is encapsulated within a shell of particles.

  9. 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.

  10. Transmembrane Protein (Perfringolysin O) Association with Ordered Membrane Domains (Rafts) Depends Upon the Raft-Associating Properties of Protein-Bound Sterol

    PubMed Central

    Lin, Qingqing; London, Erwin

    2013-01-01

    Because transmembrane (TM) protein localization, or nonlocalization, in ordered membrane domains (rafts) is a key to understanding membrane domain function, it is important to define the origin of protein-raft interaction. One hypothesis is that a tight noncovalent attachment of TM proteins to lipids that have a strong affinity for ordered domains can be sufficient to induce raft-protein interaction. The sterol-binding protein perfringolysin O (PFO) was used to test this hypothesis. PFO binds both to sterols that tend to localize in ordered domains (e.g., cholesterol), and to those that do not (e.g., coprostanol), but it does not bind to epicholesterol, a raft-promoting 3α-OH sterol. Using a fluorescence resonance energy transfer assay in model membrane vesicles containing coexisting ordered and disordered lipid domains, both TM and non-TM forms of PFO were found to concentrate in ordered domains in vesicles containing high and low-Tm lipids plus cholesterol or 1:1 (mol/mol) cholesterol/epicholesterol, whereas they concentrate in disordered domains in vesicles containing high-Tm and low-Tm lipids plus 1:1 (mol/mol) coprostanol/epicholesterol. Combined with previous studies this behavior indicates that TM protein association with ordered domains is dependent upon both the association of the protein-bound sterol with ordered domains and hydrophobic match between TM segments and rafts. PMID:24359745

  11. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions

    PubMed Central

    Kovacs, Erika; Harmat, Veronika; Tóth, Judit; Vértessy, Beáta G.; Módos, Károly; Kardos, József; Liliom, Károly

    2010-01-01

    Lipid-protein interactions are rarely characterized at a structural molecular level due to technical difficulties; however, the biological significance of understanding the mechanism of these interactions is outstanding. In this report, we provide mechanistic insight into the inhibitory complex formation of the lipid mediator sphingosylphosphorylcholine with calmodulin, the most central and ubiquitous regulator protein in calcium signaling. We applied crystallographic, thermodynamic, kinetic, and spectroscopic approaches using purified bovine calmodulin and bovine cerebral microsomal fraction to arrive at our conclusions. Here we present 1) a 1.6-Å resolution crystal structure of their complex, in which the sphingolipid occupies the conventional hydrophobic binding site on calmodulin; 2) a peculiar stoichiometry-dependent binding process: at low or high protein-to-lipid ratio calmodulin binds lipid micelles or a few lipid molecules in a compact globular conformation, respectively, and 3) evidence that the sphingolipid displaces calmodulin from its targets on cerebral microsomes. We have ascertained the specificity of the interaction using structurally related lipids as controls. Our observations reveal the structural basis of selective calmodulin inhibition by the sphingolipid. On the basis of the crystallographic and biophysical characterization of the calmodulin–sphingosylphosphorylcholine interaction, we propose a novel lipid-protein binding model, which might be applicable to other interactions as well.—Kovacs, E., Harmat, V., Tóth, J., Vértessy, B. G., Módos, K., Kardos, J., Liliom, K. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions. PMID:20522785

  12. Euphol from Euphorbia tirucalli Negatively Modulates TGF-β Responsiveness via TGF-β Receptor Segregation inside Membrane Rafts.

    PubMed

    Chen, Chun-Lin; Chen, Ying-Pin; Lin, Ming-Wei; Huang, Yaw-Bin; Chang, Fang-Rong; Duh, Tsai-Hui; Wu, Deng-Chyang; Wu, Wei-Chiang; Kao, Yu-Chen; Yang, Pei-Hua

    2015-01-01

    Transforming growth factor-β (TGF-β) responsiveness in cultured cells can be modulated by TGF-β partitioning between lipid raft/caveolae- and clathrin-mediated endocytosis pathways. Lipid rafts are plasma membrane microdomains with an important role in cell survival signaling, and cholesterol is necessary for the lipid rafts' structure and function. Euphol is a euphane-type triterpene alcohol that is structurally similar to cholesterol and has a wide range of pharmacological properties, including anti-inflammatory and anti-cancer effects. In the present study, euphol suppressed TGF-β signaling by inducing TGF-β receptor movement into lipid-raft microdomains and degrading TGF-β receptors. PMID:26448474

  13. Role of Lipids in Virus Replication

    PubMed Central

    Lorizate, Maier; Kräusslich, Hans-Georg

    2011-01-01

    Viruses intricately interact with and modulate cellular membranes at several stages of their replication, but much less is known about the role of viral lipids compared to proteins and nucleic acids. All animal viruses have to cross membranes for cell entry and exit, which occurs by membrane fusion (in enveloped viruses), by transient local disruption of membrane integrity, or by cell lysis. Furthermore, many viruses interact with cellular membrane compartments during their replication and often induce cytoplasmic membrane structures, in which genome replication and assembly occurs. Recent studies revealed details of membrane interaction, membrane bending, fission, and fusion for a number of viruses and unraveled the lipid composition of raft-dependent and -independent viruses. Alterations of membrane lipid composition can block viral release and entry, and certain lipids act as fusion inhibitors, suggesting a potential as antiviral drugs. Here, we review viral interactions with cellular membranes important for virus entry, cytoplasmic genome replication, and virus egress. PMID:21628428

  14. Contribution of PIP-5 kinase I{alpha} to raft-based Fc{gamma}RIIA signaling

    SciTech Connect

    Szymanska, Ewelina; Korzeniowski, Marek; Raynal, Patrick; Sobota, Andrzej; Kwiatkowska, Katarzyna

    2009-04-01

    Receptor Fc{gamma}IIA (Fc{gamma}RIIA) associates with plasma membrane rafts upon activation to trigger signaling cascades leading to actin polymerization. We examined whether compartmentalization of PI(4,5)P{sub 2} and PI(4,5)P{sub 2}-synthesizing PIP5-kinase I{alpha} to rafts contributes to Fc{gamma}RIIA signaling. A fraction of PIP5-kinase I{alpha} was detected in raft-originating detergent-resistant membranes (DRM) isolated from U937 monocytes and other cells. The DRM of U937 monocytes contained also a major fraction of PI(4,5)P{sub 2}. PIP5-kinase I{alpha} bound PI(4,5)P{sub 2}, and depletion of the lipid displaced PIP5-kinase I{alpha} from the DRM. Activation of Fc{gamma}RIIA in BHK transfectants led to recruitment of the kinase to the plasma membrane and enrichment of DRM in PI(4,5)P{sub 2}. Immunofluorescence studies revealed that in resting cells the kinase was associated with the plasma membrane, cytoplasmic vesicles and the nucleus. After Fc{gamma}RIIA activation, PIP5-kinase I{alpha} and PI(4,5)P{sub 2} co-localized transiently with the activated receptor at distinct cellular locations. Immunoelectron microscopy studies revealed that PIP5-kinase I{alpha} and PI(4,5)P{sub 2} were present at the edges of electron-dense assemblies containing activated Fc{gamma}RIIA in their core. The data suggest that activation of Fc{gamma}RIIA leads to membrane rafts coalescing into signaling platforms containing PIP5-kinase I{alpha} and PI(4,5)P{sub 2}.

  15. Untargeted Metabolomics Reveals Predominant Alterations in Lipid Metabolism Following Light Exposure in Broccoli Sprouts

    PubMed Central

    Maldini, Mariateresa; Natella, Fausta; Baima, Simona; Morelli, Giorgio; Scaccini, Cristina; Langridge, James; Astarita, Giuseppe

    2015-01-01

    The consumption of vegetables belonging to the family Brassicaceae (e.g., broccoli and cauliflower) is linked to a reduced incidence of cancer and cardiovascular diseases. The molecular composition of such plants is strongly affected by growing conditions. Here we developed an unbiased metabolomics approach to investigate the effect of light and dark exposure on the metabolome of broccoli sprouts and we applied such an approach to provide a bird’s-eye view of the overall metabolic response after light exposure. Broccoli seeds were germinated and grown hydroponically for five days in total darkness or with a light/dark photoperiod (16 h light/8 h dark cycle). We used an ultra-performance liquid-chromatography system coupled to an ion-mobility, time-of-flight mass spectrometer to profile the large array of metabolites present in the sprouts. Differences at the metabolite level between groups were analyzed using multivariate statistical analyses, including principal component analysis and correlation analysis. Altered metabolites were identified by searching publicly available and in-house databases. Metabolite pathway analyses were used to support the identification of subtle but significant changes among groups of related metabolites that may have gone unnoticed with conventional approaches. Besides the chlorophyll pathway, light exposure activated the biosynthesis and metabolism of sterol lipids, prenol lipids, and polyunsaturated lipids, which are essential for the photosynthetic machinery. Our results also revealed that light exposure increased the levels of polyketides, including flavonoids, and oxylipins, which play essential roles in the plant’s developmental processes and defense mechanism against herbivores. This study highlights the significant contribution of light exposure to the ultimate metabolic phenotype, which might affect the cellular physiology and nutritional value of broccoli sprouts. Furthermore, this study highlights the potential of an

  16. Untargeted Metabolomics Reveals Predominant Alterations in Lipid Metabolism Following Light Exposure in Broccoli Sprouts.

    PubMed

    Maldini, Mariateresa; Natella, Fausta; Baima, Simona; Morelli, Giorgio; Scaccini, Cristina; Langridge, James; Astarita, Giuseppe

    2015-01-01

    The consumption of vegetables belonging to the family Brassicaceae (e.g., broccoli and cauliflower) is linked to a reduced incidence of cancer and cardiovascular diseases. The molecular composition of such plants is strongly affected by growing conditions. Here we developed an unbiased metabolomics approach to investigate the effect of light and dark exposure on the metabolome of broccoli sprouts and we applied such an approach to provide a bird's-eye view of the overall metabolic response after light exposure. Broccoli seeds were germinated and grown hydroponically for five days in total darkness or with a light/dark photoperiod (16 h light/8 h dark cycle). We used an ultra-performance liquid-chromatography system coupled to an ion-mobility, time-of-flight mass spectrometer to profile the large array of metabolites present in the sprouts. Differences at the metabolite level between groups were analyzed using multivariate statistical analyses, including principal component analysis and correlation analysis. Altered metabolites were identified by searching publicly available and in-house databases. Metabolite pathway analyses were used to support the identification of subtle but significant changes among groups of related metabolites that may have gone unnoticed with conventional approaches. Besides the chlorophyll pathway, light exposure activated the biosynthesis and metabolism of sterol lipids, prenol lipids, and polyunsaturated lipids, which are essential for the photosynthetic machinery. Our results also revealed that light exposure increased the levels of polyketides, including flavonoids, and oxylipins, which play essential roles in the plant's developmental processes and defense mechanism against herbivores. This study highlights the significant contribution of light exposure to the ultimate metabolic phenotype, which might affect the cellular physiology and nutritional value of broccoli sprouts. Furthermore, this study highlights the potential of an

  17. Methyl substitution of a rexinoid agonist improves potency and reveals site of lipid toxicity.

    PubMed

    Atigadda, Venkatram R; Xia, Gang; Desphande, Anil; Boerma, LeeAnn J; Lobo-Ruppert, Susan; Grubbs, Clinton J; Smith, Craig D; Brouillette, Wayne J; Muccio, Donald D

    2014-06-26

    (2E,4E,6Z,8E)-8-(3',4'-Dihydro-1'(2'H)-naphthalen-1'-ylidene)-3,7-dimethyl-2,4,6-octatrienoic acid, 9cUAB30, is a selective rexinoid that displays substantial chemopreventive capacity with little toxicity. 4-Methyl-UAB30, an analogue of 9cUAB30, is a potent RXR agonist but caused increased lipid biosynthesis unlike 9cUAB30. To evaluate how methyl substitution influenced potency and lipid biosynthesis, we synthesized four 9cUAB30 homologues with methyl substitutions at the 5-, 6-, 7-, or 8-position of the tetralone ring. The syntheses and biological evaluations of these new analogues are reported here along with the X-ray crystal structures of each homologue bound to the ligand binding domain of hRXRα. We demonstrate that each homologue of 9cUAB30 is a more potent agonist, but only the 7-methyl-9cUAB30 caused severe hyperlipidemia in rats. On the basis of the X-ray crystal structures of these new rexinoids and bexarotene (Targretin) bound to hRXRα-LBD, we reveal that each rexinoid, which induced hyperlipidemia, had methyl groups that interacted with helix 7 residues of the LBD. PMID:24801499

  18. Methyl Substitution of a Rexinoid Agonist Improves Potency and Reveals Site of Lipid Toxicity

    PubMed Central

    2015-01-01

    (2E,4E,6Z,8E)-8-(3′,4′-Dihydro-1′(2′H)-naphthalen-1′-ylidene)-3,7-dimethyl-2,4,6-octatrienoic acid, 9cUAB30, is a selective rexinoid that displays substantial chemopreventive capacity with little toxicity. 4-Methyl-UAB30, an analogue of 9cUAB30, is a potent RXR agonist but caused increased lipid biosynthesis unlike 9cUAB30. To evaluate how methyl substitution influenced potency and lipid biosynthesis, we synthesized four 9cUAB30 homologues with methyl substitutions at the 5-, 6-, 7-, or 8-position of the tetralone ring. The syntheses and biological evaluations of these new analogues are reported here along with the X-ray crystal structures of each homologue bound to the ligand binding domain of hRXRα. We demonstrate that each homologue of 9cUAB30 is a more potent agonist, but only the 7-methyl-9cUAB30 caused severe hyperlipidemia in rats. On the basis of the X-ray crystal structures of these new rexinoids and bexarotene (Targretin) bound to hRXRα-LBD, we reveal that each rexinoid, which induced hyperlipidemia, had methyl groups that interacted with helix 7 residues of the LBD. PMID:24801499

  19. Lipid Interaction Networks of Peripheral Membrane Proteins Revealed by Data-Driven Micelle Docking

    PubMed Central

    Dancea, Felician; Kami, Keiichiro; Overduin, Michael

    2008-01-01

    Many signaling and trafficking proteins contain modular domains that bind reversibly to cellular membranes. The structural basis of the intermolecular interactions which mediate these membrane-targeting events remains elusive since protein-membrane complexes are not directly accessible to standard structural biology techniques. Here we report a fast protein-micelle docking methodology that yields three-dimensional model structures of proteins inserted into micelles, revealing energetically favorable orientations, convergent insertion angles, and an array of protein-lipid interactions at atomic resolution. The method is applied to two peripheral membrane proteins, the early endosome antigen 1 (EEA1) FYVE (a zinc finger domain found in the proteins Fab1, YOTB/ZK632.12, Vac1, and EEA1) and Vam7p phagocyte oxidase homology domains, which are revealed to form extensive networks of interactions with multiple phospholipid headgroups and acyl chains. The resulting structural models explain extensive published mutagenesis data and reveal novel binding determinants. The docking restraints used here were based on NMR data, but can be derived from any technique that detects insertion of protein residues into a membrane, and can be applied to virtually any peripheral membrane protein or membrane-like structure. PMID:17890395

  20. Europa Ice Rafts

    NASA Technical Reports Server (NTRS)

    1997-01-01

    This high resolution image shows the ice-rich crust of Europa, one of the moons of Jupiter. Seen here are crustal plates ranging up to 13 kilometers (8 miles) across, which have been broken apart and 'rafted' into new positions, superficially resembling the disruption of pack-ice on polar seas during spring thaws on Earth. The size and geometry of these features suggest that motion was enabled by ice-crusted water or soft ice close to the surface at the time of disruption.

    The area shown is about 34 kilometers by 42 kilometers (21 miles by 26 miles), centered at 9.4 degrees north latitude, 274 degrees west longitude, and the resolution is 54 meters (59 yards). This picture was taken by the Solid State Imaging system on board the Galileo spacecraft on February 20, 1997, from a distance of 5,340 kilometers (3,320 miles) during the spacecraft's close flyby of Europa.

    The Jet Propulsion Laboratory, Pasadena, CA, manages the mission for NASA's Office of Space Science, Washington D.C. This image and other images and data received from Galileo are posted on the World Wide Web Galileo mission home page at: http://galileo.jpl.nasa.gov.

  1. Vibrio vulnificus VvpE Stimulates IL-1β Production by the Hypomethylation of the IL-1β Promoter and NF-κB Activation via Lipid Raft-Dependent ANXA2 Recruitment and Reactive Oxygen Species Signaling in Intestinal Epithelial Cells.

    PubMed

    Lee, Sei-Jung; Jung, Young Hyun; Song, Eun Ju; Jang, Kyung Ku; Choi, Sang Ho; Han, Ho Jae

    2015-09-01

    An inflammatory response is a hallmark of necrosis evoked by bacterial pathogens. Vibrio vulnificus, VvpE, is an elastase that is responsible for tissue necrosis and inflammation; however, the molecular mechanism by which it regulates host cell death has not been characterized. In the present study, we investigate the cellular mechanism of VvpE with regard to host cell death and the inflammatory response of human intestinal epithelial (INT-407) cells. The recombinant protein (r)VvpE (50 pg/ml) caused cytotoxicity mainly via necrosis coupled with IL-1β production. The necrotic cell death induced by rVvpE is highly susceptible to the knockdown of annexin A (ANXA)2 and the sequestration of membrane cholesterol. We found that rVvpE induces the recruitment of NADPH oxidase 2 and neutrophil cytosolic factor 1 into membrane lipid rafts coupled with ANXA2 to facilitate the production of reactive oxygen species (ROS). The bacterial signaling of rVvpE through ROS production is uniquely mediated by the phosphorylation of redox-sensitive transcription factor NF-κB. The silencing of NF-κB inhibited IL-1β production during necrosis. rVvpE induced hypomethylation and region-specific transcriptional occupancy by NF-κB in the IL-1β promoter and has the ability to induce pyroptosis via NOD-, LRR-, and pyrin domain-containing 3 inflammasome. In a mouse model of V. vulnificus infection, the mutation of the vvpE gene from V. vulnificus negated the proinflammatory responses and maintained the physiological levels of the proliferation and migration of enterocytes. These results demonstrate that VvpE induces the hypomethylation of the IL-1β promoter and the transcriptional regulation of NF-κB through lipid raft-dependent ANXA2 recruitment and ROS signaling to promote IL-1β production in intestinal epithelial cells. PMID:26223656

  2. Roles of lipid metabolism in keloid development

    PubMed Central

    2013-01-01

    Keloids are common cutaneous pathological scars that are characterised by the histological accumulation of fibroblasts, collagen fibres, and clinically significant invasive growth. Although increasing lines of research on keloids have revealed genetic and environmental factors that contribute to their formation, the etiology of these scars remains unclear. Several studies have suggested the involvement of lipid metabolism, from a nutritional point of view. However, the role that lipid metabolism plays in the pathogenesis and progression of keloids has not previously been reviewed. The progress that has been made in understanding the roles of the pro- and anti-inflammatory lipid mediators in inflammation, and how they relate to the formation and progression of keloids, is also outlined. In particular, the possible relationships between mechanotransduction and lipid metabolites in keloids are explored. Mechanotransduction is the process by which physical forces are converted into biochemical signals that are then integrated into cellular responses. It is possible that lipid rafts and caveolae provide the location of lipid signaling and interactions between these signaling pathways and mechanotransduction. Moreover, interactions between lipid signaling pathway molecules and mechanotransduction molecules have been observed. A better understanding of the lipid profile changes and the functional roles lipid metabolism plays in keloids will help to identify target molecules for the development of novel interventions that can prevent, reduce, or even reverse pathological scar formation and/or progression. PMID:23634948

  3. Top-down lipidomics of low density lipoprotein reveal altered lipid profiles in advanced chronic kidney disease[S

    PubMed Central

    Reis, Ana; Rudnitskaya, Alisa; Chariyavilaskul, Pajaree; Dhaun, Neeraj; Melville, Vanessa; Goddard, Jane; Webb, David J.; Pitt, Andrew R.; Spickett, Corinne M.

    2015-01-01

    This study compared the molecular lipidomic profile of LDL in patients with nondiabetic advanced renal disease and no evidence of CVD to that of age-matched controls, with the hypothesis that it would reveal proatherogenic lipid alterations. LDL was isolated from 10 normocholesterolemic patients with stage 4/5 renal disease and 10 controls, and lipids were analyzed by accurate mass LC/MS. Top-down lipidomics analysis and manual examination of the data identified 352 lipid species, and automated comparative analysis demonstrated alterations in lipid profile in disease. The total lipid and cholesterol content was unchanged, but levels of triacylglycerides and N-acyltaurines were significantly increased, while phosphatidylcholines, plasmenyl ethanolamines, sulfatides, ceramides, and cholesterol sulfate were significantly decreased in chronic kidney disease (CKD) patients. Chemometric analysis of individual lipid species showed very good discrimination of control and disease sample despite the small cohorts and identified individual unsaturated phospholipids and triglycerides mainly responsible for the discrimination. These findings illustrate the point that although the clinical biochemistry parameters may not appear abnormal, there may be important underlying lipidomic changes that contribute to disease pathology. The lipidomic profile of CKD LDL offers potential for new biomarkers and novel insights into lipid metabolism and cardiovascular risk in this disease. PMID:25424003

  4. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions.

    PubMed

    Kovacs, Erika; Harmat, Veronika; Tóth, Judit; Vértessy, Beáta G; Módos, Károly; Kardos, József; Liliom, Károly

    2010-10-01

    Lipid-protein interactions are rarely characterized at a structural molecular level due to technical difficulties; however, the biological significance of understanding the mechanism of these interactions is outstanding. In this report, we provide mechanistic insight into the inhibitory complex formation of the lipid mediator sphingosylphosphorylcholine with calmodulin, the most central and ubiquitous regulator protein in calcium signaling. We applied crystallographic, thermodynamic, kinetic, and spectroscopic approaches using purified bovine calmodulin and bovine cerebral microsomal fraction to arrive at our conclusions. Here we present 1) a 1.6-Å resolution crystal structure of their complex, in which the sphingolipid occupies the conventional hydrophobic binding site on calmodulin; 2) a peculiar stoichiometry-dependent binding process: at low or high protein-to-lipid ratio calmodulin binds lipid micelles or a few lipid molecules in a compact globular conformation, respectively, and 3) evidence that the sphingolipid displaces calmodulin from its targets on cerebral microsomes. We have ascertained the specificity of the interaction using structurally related lipids as controls. Our observations reveal the structural basis of selective calmodulin inhibition by the sphingolipid. On the basis of the crystallographic and biophysical characterization of the calmodulin-sphingosylphosphorylcholine interaction, we propose a novel lipid-protein binding model, which might be applicable to other interactions as well. PMID:20522785

  5. Interleukin-1-induced gene expression requires the membrane-raft-dependent internalization of the interleukin-1 receptor.

    PubMed

    Windheim, Mark

    2016-10-01

    Interleukin-1 (IL-1) binding to its receptor triggers signaling events at the plasma membrane that are essential but not sufficient for the induction of the IL-1-dependent gene expression. In addition, the ligand-induced endocytosis of the IL-1 receptor and signaling events that are initiated after the internalization of the IL-1 receptor presumably involving signaling endosomes are critical for the IL-1-induced gene expression. In this study, we investigate the role of membrane domains, commonly denoted as lipid rafts, in the IL-1-induced signal transduction. We demonstrate that the internalization of the IL-1 receptor depends on the integrity of lipid rafts and that the disruption of lipid rafts strongly reduces the IL-1-induced gene expression. Interestingly, the IL-1-dependent signaling events activated at the plasma membrane are not influenced by the disruption of lipid rafts suggesting that IL-1 signaling is initiated in a non-raft domain of the plasma membrane. Subsequently, the IL-1 receptor is translocated to lipid rafts where receptor endocytosis occurs to enable the internalization-dependent IL-1 signaling to activate the IL-1-induced gene expression. PMID:27327966

  6. The Combining Sites of Anti-lipid A Antibodies Reveal a Widely Utilized Motif Specific for Negatively Charged Groups.

    PubMed

    Haji-Ghassemi, Omid; Müller-Loennies, Sven; Rodriguez, Teresa; Brade, Lore; Grimmecke, Hans-Dieter; Brade, Helmut; Evans, Stephen V

    2016-05-01

    Lipopolysaccharide dispersed in the blood by Gram-negative bacteria can be a potent inducer of septic shock. One research focus has been based on antibody sequestration of lipid A (the endotoxic principle of LPS); however, none have been successfully developed into a clinical treatment. Comparison of a panel of anti-lipid A antibodies reveals highly specific antibodies produced through distinct germ line precursors. The structures of antigen-binding fragments for two homologous mAbs specific for lipid A, S55-3 and S55-5, have been determined both in complex with lipid A disaccharide backbone and unliganded. These high resolution structures reveal a conserved positively charged pocket formed within the complementarity determining region H2 loops that binds the terminal phosphates of lipid A. Significantly, this motif occurs in unrelated antibodies where it mediates binding to negatively charged moieties through a range of epitopes, including phosphorylated peptides used in diagnostics and therapeutics. S55-3 and S55-5 have combining sites distinct from anti-lipid A antibodies previously described (as a result of their separate germ line origin), which are nevertheless complementary both in shape and charge to the antigen. S55-3 and S55-5 display similar avidity toward lipid A despite possessing a number of different amino acid residues in their combining sites. Binding of lipid A occurs independent of the acyl chains, although the GlcN-O6 attachment point for the core oligosaccharide is buried in the combining site, which explains their inability to recognize LPS. Despite their lack of therapeutic potential, the observed motif may have significant immunological implications as a tool for engineering recombinant antibodies. PMID:26933033

  7. High Molecular Weight Complex Analysis of Epstein-Barr Virus Latent Membrane Protein 1 (LMP-1): Structural Insights into LMP-1’s Homo-Oligomerization and Lipid Raft Association

    PubMed Central

    Wrobel, Christopher M.; Geiger, Timothy R.; Nix, Rebecca N.; Robitaille, Aaron M.; Balser, Sandra; Cervantes, Alfredo; Gonzalez, Miguel; Martin, Jennifer M.

    2013-01-01

    LMP-1 is a constitutively active Tumor Necrosis Factor Receptor analog encoded by Epstein-Barr virus. LMP-1 activation correlates with oligomerization and raft localization, but direct evidence of LMP-1 oligomers is limited. We report that LMP-1 forms multiple high molecular weight native LMP-1 complexes when analyzed by BN-PAGE, the largest of which are enriched in detergent resistant membranes. The largest of these high molecular weight complexes are not formed by purified LMP-1 or by loss of function LMP-1 mutants. Consistent with these results we find a dimeric form of LMP-1 that can be stabilized by disulfide crosslinking. We identify cysteine 238 in the C-terminus of LMP-1 as the crosslinked cysteine. Disulfide crosslinking occurs post-lysis but the dimer can be crosslinked in intact cells with membrane permeable crosslinkers. LMP-1/C238A retains wild type LMP-1 NF-κB activity. LMP-1’s TRAF binding, raft association and oligomerization are associated with the dimeric form of LMP-1. Our results suggest the possibility that the observed dimeric species results from inter-oligomeric crosslinking of LMP-1 molecules in adjacent core LMP-1 oligomers. PMID:24075898

  8. TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action.

    PubMed

    Gao, Yuan; Cao, Erhu; Julius, David; Cheng, Yifan

    2016-06-16

    When integral membrane proteins are visualized in detergents or other artificial systems, an important layer of information is lost regarding lipid interactions and their effects on protein structure. This is especially relevant to proteins for which lipids have both structural and regulatory roles. Here we demonstrate the power of combining electron cryo-microscopy with lipid nanodisc technology to ascertain the structure of the rat TRPV1 ion channel in a native bilayer environment. Using this approach, we determined the locations of annular and regulatory lipids and showed that specific phospholipid interactions enhance binding of a spider toxin to TRPV1 through formation of a tripartite complex. Furthermore, phosphatidylinositol lipids occupy the binding site for capsaicin and other vanilloid ligands, suggesting a mechanism whereby chemical or thermal stimuli elicit channel activation by promoting the release of bioactive lipids from a critical allosteric regulatory site. PMID:27281200

  9. MALDI imaging MS reveals candidate lipid markers of polycystic kidney disease.

    PubMed

    Ruh, Hermelindis; Salonikios, Theresia; Fuchser, Jens; Schwartz, Matthias; Sticht, Carsten; Hochheim, Christina; Wirnitzer, Bernhard; Gretz, Norbert; Hopf, Carsten

    2013-10-01

    Autosomal recessive polycystic kidney disease (ARPKD) is a severe, monogenetically inherited kidney and liver disease. PCK rats carrying the orthologous mutant gene serve as a model of human disease, and alterations in lipid profiles in PCK rats suggest that defined subsets of lipids may be useful as molecular disease markers. Whereas MALDI protein imaging mass spectrometry (IMS) has become a promising tool for disease classification, widely applicable workflows that link MALDI lipid imaging and identification as well as structural characterization of candidate disease-classifying marker lipids are lacking. Here, we combine selective MALDI imaging of sulfated kidney lipids and Fisher discriminant analysis (FDA) of imaging data sets for identification of candidate markers of progressive disease in PCK rats. Our study highlights strong increases in lower mass lipids as main classifiers of cystic disease. Structure determination by high-resolution mass spectrometry identifies these altered lipids as taurine-conjugated bile acids. These sulfated lipids are selectively elevated in the PCK rat model but not in models of related hepatorenal fibrocystic diseases, suggesting that they be molecular markers of the disease and that a combination of MALDI imaging with high-resolution MS methods and Fisher discriminant data analysis may be applicable for lipid marker discovery. PMID:23852700

  10. Metabolomic analysis reveals mechanism of antioxidant butylated hydroxyanisole on lipid accumulation in Crypthecodinium cohnii.

    PubMed

    Sui, Xiao; Niu, Xiangfeng; Shi, Mengliang; Pei, Guangsheng; Li, Jinghan; Chen, Lei; Wang, Jiangxin; Zhang, Weiwen

    2014-12-24

    The heterotrophic dinoflagellate alga Crypthecodinium cohnii is known to accumulate lipids with a high fraction of docosahexaenoic acid (DHA). In this study, we first evaluated two antioxidant compounds, butylated hydroxyanisole (BHA) and propyl gallate (PG), for their effects on lipid accumulation in C. cohnii. The results showed that antioxidant BHA could increase lipid accumulation in C. cohnii by 8.80% at a final concentration of 30 μM, while PG had no obvious effect on lipid accumulation at the tested concentrations. To decipher the molecular mechanism responsible for the increased lipid accumulation by BHA, we employed an integrated GC-MS and LC-MS metabolomic approach to determine the time-series metabolic profiles with or without BHA, and then subjected the metabolomic data to a principal component analysis (PCA) and a weighted gene coexpression network analysis (WGCNA) network analyses to identify the key metabolic modules and metabolites possibly relevant to the increased lipid accumulation. LC-MS analysis showed that several metabolites, including NADPH, could be important for the stimulation role of BHA on lipid accumulation. Meanwhile GC-MS and network analyses allowed identification of eight metabolic modules and nine hub metabolites possibly relevant to the stimulation role of BHA in C. cohnii. The study provided a metabolomics view of the BHA mode of action on lipid accumulation in C. cohnii, and the information could be valuable for a better understanding of antioxidant effects on lipid accumulation in other microalgae as well. PMID:25436856

  11. Desorption Electrospray Ionization Mass Spectrometry Reveals Lipid Metabolism of Individual Oocytes and Embryos

    PubMed Central

    González-Serrano, Andrés Felipe; Pirro, Valentina; Ferreira, Christina R.; Oliveri, Paolo; Eberlin, Livia S.; Heinzmann, Julia; Lucas-Hahn, Andrea; Niemann, Heiner; Cooks, Robert Graham

    2013-01-01

    Alteration of maternal lipid metabolism early in development has been shown to trigger obesity, insulin resistance, type 2 diabetes and cardiovascular diseases later in life in humans and animal models. Here, we set out to determine (i) lipid composition dynamics in single oocytes and preimplantation embryos by high mass resolution desorption electrospray ionization mass spectrometry (DESI-MS), using the bovine species as biological model, (ii) the metabolically most relevant lipid compounds by multivariate data analysis and (iii) lipid upstream metabolism by quantitative real-time PCR (qRT-PCR) analysis of several target genes (ACAT1, CPT 1b, FASN, SREBP1 and SCAP). Bovine oocytes and blastocysts were individually analyzed by DESI-MS in both positive and negative ion modes, without lipid extraction and under ambient conditions, and were profiled for free fatty acids (FFA), phospholipids (PL), cholesterol-related molecules, and triacylglycerols (TAG). Principal component analysis (PCA) and linear discriminant analysis (LDA), performed for the first time on DESI-MS fused data, allowed unequivocal discrimination between oocytes and blastocysts based on specific lipid profiles. This analytical approach resulted in broad and detailed lipid annotation of single oocytes and blastocysts. Results of DESI-MS and transcript regulation analysis demonstrate that blastocysts produced in vitro and their in vivo counterparts differed significantly in the homeostasis of cholesterol and FFA metabolism. These results should assist in the production of viable and healthy embryos by elucidating in vivo embryonic lipid metabolism. PMID:24073231

  12. Lipidomics reveals control of Mycobacterium tuberculosis virulence lipids via metabolic coupling.

    PubMed

    Jain, Madhulika; Petzold, Christopher J; Schelle, Michael W; Leavell, Michael D; Mougous, Joseph D; Bertozzi, Carolyn R; Leary, Julie A; Cox, Jeffery S

    2007-03-20

    Mycobacterium tuberculosis synthesizes specific polyketide lipids that interact with the host and are required for virulence. Using a mass spectrometric approach to simultaneously monitor hundreds of lipids, we discovered that the size and abundance of two lipid virulence factors, phthiocerol dimycocerosate (PDIM) and sulfolipid-1 (SL-1), are controlled by the availability of a common precursor, methyl malonyl CoA (MMCoA). Consistent with this view, increased levels of MMCoA led to increased abundance and mass of both PDIM and SL-1. Furthermore, perturbation of MMCoA metabolism attenuated pathogen replication in mice. Importantly, we detected increased PDIM synthesis in bacteria growing within host tissues and in bacteria grown in culture on odd-chain fatty acids. Because M. tuberculosis catabolizes host lipids to grow during infection, we propose that growth of M. tuberculosis on fatty acids in vivo leads to increased flux of MMCoA through lipid biosynthetic pathways, resulting in increased virulence lipid synthesis. Our results suggest that the shift to host lipid catabolism during infection allows for increased virulence lipid anabolism by the bacterium. PMID:17360366

  13. Dysregulated signaling hubs of liver lipid metabolism reveal hepatocellular carcinoma pathogenesis.

    PubMed

    Lee, Sunjae; Mardinoglu, Adil; Zhang, Cheng; Lee, Doheon; Nielsen, Jens

    2016-07-01

    Hepatocellular carcinoma (HCC) has a high mortality rate and early detection of HCC is crucial for the application of effective treatment strategies. HCC is typically caused by either viral hepatitis infection or by fatty liver disease. To diagnose and treat HCC it is necessary to elucidate the underlying molecular mechanisms. As a major cause for development of HCC is fatty liver disease, we here investigated anomalies in regulation of lipid metabolism in the liver. We applied a tailored network-based approach to identify signaling hubs associated with regulation of this part of metabolism. Using transcriptomics data of HCC patients, we identified significant dysregulated expressions of lipid-regulated genes, across many different lipid metabolic pathways. Our findings, however, show that viral hepatitis causes HCC by a distinct mechanism, less likely involving lipid anomalies. Based on our analysis we suggest signaling hub genes governing overall catabolic or anabolic pathways, as novel drug targets for treatment of HCC that involves lipid anomalies. PMID:27216817

  14. Dysregulated signaling hubs of liver lipid metabolism reveal hepatocellular carcinoma pathogenesis

    PubMed Central

    Lee, Sunjae; Mardinoglu, Adil; Zhang, Cheng; Lee, Doheon; Nielsen, Jens

    2016-01-01

    Hepatocellular carcinoma (HCC) has a high mortality rate and early detection of HCC is crucial for the application of effective treatment strategies. HCC is typically caused by either viral hepatitis infection or by fatty liver disease. To diagnose and treat HCC it is necessary to elucidate the underlying molecular mechanisms. As a major cause for development of HCC is fatty liver disease, we here investigated anomalies in regulation of lipid metabolism in the liver. We applied a tailored network-based approach to identify signaling hubs associated with regulation of this part of metabolism. Using transcriptomics data of HCC patients, we identified significant dysregulated expressions of lipid-regulated genes, across many different lipid metabolic pathways. Our findings, however, show that viral hepatitis causes HCC by a distinct mechanism, less likely involving lipid anomalies. Based on our analysis we suggest signaling hub genes governing overall catabolic or anabolic pathways, as novel drug targets for treatment of HCC that involves lipid anomalies. PMID:27216817

  15. Targeting Acetylcholinesterase to Membrane Rafts

    PubMed Central

    Xie, Heidi Q.; Liang, Dong; Leung, K. Wing; Chen, Vicky P.; Zhu, Kevin Y.; Chan, Wallace K. B.; Choi, Roy C. Y.; Massoulié, Jean; Tsim, Karl W. K.

    2010-01-01

    In the mammalian brain, acetylcholinesterase (AChE) is anchored in cell membranes by a transmembrane protein PRiMA (proline-rich membrane anchor). We present evidence that at least part of the PRiMA-linked AChE is integrated in membrane microdomains called rafts. A significant proportion of PRiMA-linked AChE tetramers from rat brain was recovered in raft fractions; this proportion was markedly higher at low rather than at high concentrations of cold Triton X-100. The detergent-resistant fraction increased during brain development. In NG108-15 neuroblastoma cells transfected with cDNAs encoding AChET and PRiMA, PRiMA-linked G4 AChE was found in membrane rafts and showed the same sensitivity to cold Triton X-100 extraction as in the brain. The association of PRiMA-linked AChE with rafts was weaker than that of glycosylphosphatidylinositol-anchored G2 AChE or G4 QN-HC-linked AChE. It was found to depend on the presence of a cholesterol-binding motif, called CRAC (cholesterol recognition/interaction amino acid consensus), located at the junction of transmembrane and cytoplasmic domains of both PRiMA I and II isoforms. The cytoplasmic domain of PRiMA, which differs between PRiMA I and PRiMA II, appeared to play some role in stabilizing the raft localization of G4 AChE, because the Triton X-100-resistant fraction was smaller with the shorter PRiMA II isoform than that with the longer PRiMA I isoform. PMID:20147288

  16. New Insights into Nisin's Antibacterial Mechanism Revealed by Binding Studies with Synthetic Lipid II Analogues.

    PubMed

    't Hart, Peter; Oppedijk, Sabine F; Breukink, Eefjan; Martin, Nathaniel I

    2016-01-12

    Nisin is the preeminent lantibiotic, and to date its antibacterial mechanism has been investigated using a variety of techniques. While nisin's lipid II-mediated mode of action is well-established, a detailed analysis of the thermodynamic parameters governing this interaction has not been previously reported. We here describe an approach employing isothermal titration calorimetry to directly measure the affinity of nisin for lipid II and a number of synthetic lipid II precursors and analogues. Our measurements confirm the pyrophosphate unit of lipid II as the primary site of nisin binding and also indicate that the complete MurNAc moiety is required for a high-affinity interaction. Additionally, we find that while the pentapeptide unit of the lipid II molecule is not required for strong binding by nisin, it does play an important role in stabilizing the subsequently formed nisin-lipid II pore complex, albeit at an entropic cost. The anchoring of lipid II in a membrane environment was also found to play a significant role in enhancing nisin binding and is required in order to achieve a high-affinity interaction. PMID:26653142

  17. Reorganization of Lipid Diffusion by Myelin Basic Protein as Revealed by STED Nanoscopy.

    PubMed

    Steshenko, Olena; Andrade, Débora M; Honigmann, Alf; Mueller, Veronika; Schneider, Falk; Sezgin, Erdinc; Hell, Stefan W; Simons, Mikael; Eggeling, Christian

    2016-06-01

    Myelin is a multilayered membrane that ensheathes axonal fibers in the vertebrate nervous system, allowing fast propagation of nerve action potentials. It contains densely packed lipids, lacks an actin-based cytocortex, and requires myelin basic protein (MBP) as its major structural component. This protein is the basic constituent of the proteinaceous meshwork that is localized between adjacent cytoplasmic membranes of the myelin sheath. Yet, it is not clear how MBP influences the organization and dynamics of the lipid constituents of myelin. Here, we used optical stimulated emission depletion super-resolution microscopy in combination with fluorescence correlation spectroscopy to assess the characteristics of diffusion of different fluorescent lipid analogs in myelin membrane sheets of cultured oligodendrocytes and in micrometer-sized domains that were induced by MBP in live epithelial PtK2 cells. Lipid diffusion was significantly faster and less anomalous both in oligodendrocytes and inside the MBP-rich domains of PtK2 cells compared with undisturbed live PtK2 cells. Our data show that MBP reorganizes lipid diffusion, possibly by preventing the buildup of an actin-based cytocortex and by preventing most membrane proteins from entering the myelin sheath region. Yet, in contrast to myelin sheets in oligodendrocytes, the MBP-induced domains in epithelial PtK2 cells demonstrate no change in lipid order, indicating that segregation of long-chain lipids into myelin sheets is a process specific to oligodendrocytes. PMID:27276262

  18. Human Haploid Cell Genetics Reveals Roles for Lipid Metabolism Genes in Nonapoptotic Cell Death

    PubMed Central

    2016-01-01

    Little is known about the regulation of nonapoptotic cell death. Using massive insertional mutagenesis of haploid KBM7 cells we identified nine genes involved in small-molecule-induced nonapoptotic cell death, including mediators of fatty acid metabolism (ACSL4) and lipid remodeling (LPCAT3) in ferroptosis. One novel compound, CIL56, triggered cell death dependent upon the rate-limiting de novo lipid synthetic enzyme ACC1. These results provide insight into the genetic regulation of cell death and highlight the central role of lipid metabolism in nonapoptotic cell death. PMID:25965523

  19. Metabolomic profiling reveals mitochondrial-derived lipid biomarkers that drive obesity-associated inflammation.

    PubMed

    Sampey, Brante P; Freemerman, Alex J; Zhang, Jimmy; Kuan, Pei-Fen; Galanko, Joseph A; O'Connell, Thomas M; Ilkayeva, Olga R; Muehlbauer, Michael J; Stevens, Robert D; Newgard, Christopher B; Brauer, Heather A; Troester, Melissa A; Makowski, Liza

    2012-01-01

    Obesity has reached epidemic proportions worldwide. Several animal models of obesity exist, but studies are lacking that compare traditional lard-based high fat diets (HFD) to "Cafeteria diets" (CAF) consisting of nutrient poor human junk food. Our previous work demonstrated the rapid and severe obesogenic and inflammatory consequences of CAF compared to HFD including rapid weight gain, markers of Metabolic Syndrome, multi-tissue lipid accumulation, and dramatic inflammation. To identify potential mediators of CAF-induced obesity and Metabolic Syndrome, we used metabolomic analysis to profile serum, muscle, and white adipose from rats fed CAF, HFD, or standard control diets. Principle component analysis identified elevations in clusters of fatty acids and acylcarnitines. These increases in metabolites were associated with systemic mitochondrial dysfunction that paralleled weight gain, physiologic measures of Metabolic Syndrome, and tissue inflammation in CAF-fed rats. Spearman pairwise correlations between metabolites, physiologic, and histologic findings revealed strong correlations between elevated markers of inflammation in CAF-fed animals, measured as crown like structures in adipose, and specifically the pro-inflammatory saturated fatty acids and oxidation intermediates laurate and lauroyl carnitine. Treatment of bone marrow-derived macrophages with lauroyl carnitine polarized macrophages towards the M1 pro-inflammatory phenotype through downregulation of AMPK and secretion of pro-inflammatory cytokines. Results presented herein demonstrate that compared to a traditional HFD model, the CAF diet provides a robust model for diet-induced human obesity, which models Metabolic Syndrome-related mitochondrial dysfunction in serum, muscle, and adipose, along with pro-inflammatory metabolite alterations. These data also suggest that modifying the availability or metabolism of saturated fatty acids may limit the inflammation associated with obesity leading to Metabolic

  20. Euphol from Euphorbia tirucalli Negatively Modulates TGF-β Responsiveness via TGF-β Receptor Segregation inside Membrane Rafts

    PubMed Central

    Chen, Chun-Lin; Chen, Ying-Pin; Lin, Ming-Wei; Huang, Yaw-Bin; Chang, Fang-Rong; Duh, Tsai-Hui; Wu, Deng-Chyang; Wu, Wei-Chiang; Kao, Yu-Chen; Yang, Pei-Hua

    2015-01-01

    Transforming growth factor-β (TGF-β) responsiveness in cultured cells can be modulated by TGF-β partitioning between lipid raft/caveolae- and clathrin-mediated endocytosis pathways. Lipid rafts are plasma membrane microdomains with an important role in cell survival signaling, and cholesterol is necessary for the lipid rafts’ structure and function. Euphol is a euphane-type triterpene alcohol that is structurally similar to cholesterol and has a wide range of pharmacological properties, including anti-inflammatory and anti-cancer effects. In the present study, euphol suppressed TGF-β signaling by inducing TGF-β receptor movement into lipid-raft microdomains and degrading TGF-β receptors. PMID:26448474

  1. A Model of Lipid-Free Apolipoprotein A-I Revealed by Iterative Molecular Dynamics Simulation

    PubMed Central

    Zhang, Xing; Lei, Dongsheng; Zhang, Lei; Rames, Matthew; Zhang, Shengli

    2015-01-01

    Apolipoprotein A-I (apo A-I), the major protein component of high-density lipoprotein, has been proven inversely correlated to cardiovascular risk in past decades. The lipid-free state of apo A-I is the initial stage which binds to lipids forming high-density lipoprotein. Molecular models of lipid-free apo A-I have been reported by methods like X-ray crystallography and chemical cross-linking/mass spectrometry (CCL/MS). Through structural analysis we found that those current models had limited consistency with other experimental results, such as those from hydrogen exchange with mass spectrometry. Through molecular dynamics simulations, we also found those models could not reach a stable equilibrium state. Therefore, by integrating various experimental results, we proposed a new structural model for lipid-free apo A-I, which contains a bundled four-helix N-terminal domain (1–192) that forms a variable hydrophobic groove and a mobile short hairpin C-terminal domain (193–243). This model exhibits an equilibrium state through molecular dynamics simulation and is consistent with most of the experimental results known from CCL/MS on lysine pairs, fluorescence resonance energy transfer and hydrogen exchange. This solution-state lipid-free apo A-I model may elucidate the possible conformational transitions of apo A-I binding with lipids in high-density lipoprotein formation. PMID:25793886

  2. The cholesterol-dependent cytolysin listeriolysin O aggregates rafts via oligomerization.

    PubMed

    Gekara, Nelson O; Jacobs, Thomas; Chakraborty, Trinad; Weiss, Siegfried

    2005-09-01

    The pore-forming toxin listeriolysin O (LLO) is the main virulence factor of Listeria monocytogenes. LLO is known to act as a pseudo cytokine/chemokine, which induces a broad spectrum of host responses that ultimately influences the outcome of listeriosis. In the present study we demonstrate that LLO is a potent aggregator of lipid rafts. LLO was found to aggregate the raft associated molecules GM1, the GPI-anchored proteins CD14 and CD16 as well as the tyrosine kinase Lyn. Abrogation of the cytolytic activity of LLO by cholesterol pretreatment was found not to interfere with LLO's ability to aggregate rafts or trigger tyrosine phosphorylation in cells. However, a monoclonal antibody that blocks the oligomerization of LLO was found to inhibit rafts' aggregation as well as the induction of tyrosine phosphorylation. This implies that rafts aggregation by LLO which is independent of cytolytic activity, is due to the oligomerization of its membrane bound toxin monomers. Thus, LLO most likely induces signalling through the coaggregation of rafts' associated receptors, kinases and adaptors. PMID:16098221

  3. A model of lipid-free Apolipoprotein A-I revealed by iterative molecular dynamics simulation

    DOE PAGESBeta

    Zhang, Xing; Lei, Dongsheng; Zhang, Lei; Rames, Matthew; Zhang, Shengli

    2015-03-20

    Apolipoprotein A-I (apo A-I), the major protein component of high-density lipoprotein, has been proven inversely correlated to cardiovascular risk in past decades. The lipid-free state of apo A-I is the initial stage which binds to lipids forming high-density lipoprotein. Molecular models of lipid-free apo A-I have been reported by methods like X-ray crystallography and chemical cross-linking/mass spectrometry (CCL/MS). Through structural analysis we found that those current models had limited consistency with other experimental results, such as those from hydrogen exchange with mass spectrometry. Through molecular dynamics simulations, we also found those models could not reach a stable equilibrium state. Therefore,more » by integrating various experimental results, we proposed a new structural model for lipidfree apo A-I, which contains a bundled four-helix N-terminal domain (1–192) that forms a variable hydrophobic groove and a mobile short hairpin C-terminal domain (193–243). This model exhibits an equilibrium state through molecular dynamics simulation and is consistent with most of the experimental results known from CCL/MS on lysine pairs, fluorescence resonance energy transfer and hydrogen exchange. This solution-state lipid-free apo A-I model may elucidate the possible conformational transitions of apo A-I binding with lipids in high-density lipoprotein formation.« less

  4. A model of lipid-free Apolipoprotein A-I revealed by iterative molecular dynamics simulation

    SciTech Connect

    Zhang, Xing; Lei, Dongsheng; Zhang, Lei; Rames, Matthew; Zhang, Shengli

    2015-03-20

    Apolipoprotein A-I (apo A-I), the major protein component of high-density lipoprotein, has been proven inversely correlated to cardiovascular risk in past decades. The lipid-free state of apo A-I is the initial stage which binds to lipids forming high-density lipoprotein. Molecular models of lipid-free apo A-I have been reported by methods like X-ray crystallography and chemical cross-linking/mass spectrometry (CCL/MS). Through structural analysis we found that those current models had limited consistency with other experimental results, such as those from hydrogen exchange with mass spectrometry. Through molecular dynamics simulations, we also found those models could not reach a stable equilibrium state. Therefore, by integrating various experimental results, we proposed a new structural model for lipidfree apo A-I, which contains a bundled four-helix N-terminal domain (1–192) that forms a variable hydrophobic groove and a mobile short hairpin C-terminal domain (193–243). This model exhibits an equilibrium state through molecular dynamics simulation and is consistent with most of the experimental results known from CCL/MS on lysine pairs, fluorescence resonance energy transfer and hydrogen exchange. This solution-state lipid-free apo A-I model may elucidate the possible conformational transitions of apo A-I binding with lipids in high-density lipoprotein formation.

  5. Raft River geoscience case study

    SciTech Connect

    Dolenc, M.R.; Hull, L.C.; Mizell, S.A.; Russell, B.F.; Skiba, P.A.; Strawn, J.A.; Tullis, J.A.

    1981-11-01

    The Raft River Geothermal Site has been evaluated over the past eight years by the United States Geological Survey and the Idaho National Engineering Laboratory as a moderate-temperature geothermal resource. The geoscience data gathered in the drilling and testing of seven geothermal wells suggest that the Raft River thermal reservoir is: (a) produced from fractures found at the contact metamorphic zone, apparently the base of detached normal faulting from the Bridge and Horse Well Fault zones of the Jim Sage Mountains; (b) anisotropic, with the major axis of hydraulic conductivity coincident to the Bridge Fault Zone; (c) hydraulically connected to the shallow thermal fluid of the Crook and BLM wells based upon both geochemistry and pressure response; (d) controlled by a mixture of diluted meteoric water recharging from the northwest and a saline sodium chloride water entering from the southwest. Although the hydrogeologic environment of the Raft River geothermal area is very complex and unique, it is typical of many Basin and Range systems.

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

    PubMed Central

    Gungor, Burcin; Gombos, Imre; Crul, Tim; Ayaydin, Ferhan; Szabó, László; Török, Zsolt; Mátés, Lajos; Vígh, László; Horváth, Ibolya

    2014-01-01

    Eukaryotic cells exhibit a characteristic response to hyperthermic treatment, involving morphological and cytoskeletal alterations and the induction of heat shock protein synthesis. Small GTPases of the Ras superfamily are known to serve as molecular switches which mediate responses to extracellular stimuli. We addressed here how small GTPase Rac1 integrates signals from heat stress and simultaneously induces various cellular changes in mammalian cells. As evidence that Rac1 is implicated in the heat shock response, we first demonstrated that both mild (41.5°C) and severe (43°C) heat shock induced membrane translocation of Rac1. Following inhibition of the activation or palmitoylation of Rac1, the size of its plasma membrane-bound pool was significantly decreased while the heat shock-induced alterations in the cytoskeleton and cell morphology were prevented. We earlier documented that the size distribution pattern of cholesterol-rich rafts is temperature dependent and hypothesized that this is coupled to the triggering mechanism of stress sensing and signaling. Interestingly, when plasma membrane localization of Rac1 was inhibited, a different and temperature independent average domain size was detected. In addition, inhibition of the activation or palmitoylation of Rac1 resulted in a strongly decreased expression of the genes of major heat shock proteins hsp25 and hsp70 under both mild and severe heat stress conditions. PMID:24586549

  7. Gene expression profiling reveals Nef induced deregulation of lipid metabolism in HIV-1 infected T cells.

    PubMed

    Shrivastava, Surya; Trivedi, Jay; Mitra, Debashis

    2016-03-25

    Human Immunodeficiency Virus-1 (HIV-1) encodes a 27 kDa Negative Factor or Nef protein, which is increasingly proving to be a misnomer. Nef seems to be crucial for AIDS progression as individuals infected with nef-deleted strain of HIV were reported to become Long Term Non Progressors (LTNP). These findings necessitate tracing of Nef's footprint on landscape of cellular transcriptome favoring HIV-1 pathogenesis. We have tried to explore effect of Nef on cellular gene expression profile in conjunction with rest of HIV-1 proteins. Our results show that 237 genes are differentially regulated due to the presence of Nef during infection, which belong to several broad categories like "signaling", "apoptosis", "transcription" and "lipid metabolism" in gene ontology analysis. Furthermore, our results show that Nef causes disruption of lipid content in HIV-1 infected T cells. Molecular inhibitors of lipid metabolism like Atorvastatin and Ranolazine were found to have profound effect on wild type virus as compared to nef-deleted HIV-1. Thus our results suggest that interference in lipid metabolism is a potential mechanism through which Nef contributes in enhancing HIV-1 pathogenesis. PMID:26915805

  8. Lipidomic analysis of lipid droplets from murine hepatocytes reveals distinct signatures for nutritional stress.

    PubMed

    Chitraju, Chandramohan; Trötzmüller, Martin; Hartler, Jürgen; Wolinski, Heimo; Thallinger, Gerhard G; Lass, Achim; Zechner, Rudolf; Zimmermann, Robert; Köfeler, Harald C; Spener, Friedrich

    2012-10-01

    Liver steatosis can be induced by fasting or high-fat diet. We investigated by lipidomic analysis whether such metabolic states are reflected in the lipidome of hepatocyte lipid droplets (LDs) from mice fed normal chow diet (FED), fasted (FAS), or fed a high-fat diet (HFD). LC-MS/MS at levels of lipid species profiles and of lipid molecular species uncovered a FAS phenotype of LD enriched in triacylglycerol (TG) molecular species with very long-chain (VLC)-PUFA residues and an HFD phenotype with less unsaturated TG species in addition to characteristic lipid marker species. Nutritional stress did not result in dramatic structural alterations in diacylglycerol (DG) and phospholipid (PL) classes. Moreover, molecular species of bulk TG and of DG indicated concomitant de novo TG synthesis and lipase-catalyzed degradation to be active in LDs. DG species with VLC-PUFA residues would be preferred precursors for phosphatidylcholine (PC) species, the others for TG molecular species. In addition, molecular species of PL classes fitted the hepatocyte Kennedy and phosphatidylethanolamine methyltransferase pathways. We demonstrate that lipidomic analysis of LDs enables phenotyping of nutritional stress. TG species are best suited for such phenotyping, whereas structural analysis of TG, DG, and PL molecular species provides metabolic insights. PMID:22872753

  9. Unprecedented staining of polar lipids by a luminescent rhenium complex revealed by FTIR microspectroscopy in adipocytes.

    PubMed

    Bader, C A; Carter, E A; Safitri, A; Simpson, P V; Wright, P; Stagni, S; Massi, M; Lay, P A; Brooks, D A; Plush, S E

    2016-06-21

    Fourier transform infrared (FTIR) microspectroscopy and confocal imaging have been used to demonstrate that the neutral rhenium(i) tricarbonyl 1,10-phenanthroline complex bound to 4-cyanophenyltetrazolate as the ancillary ligand is able to localise in regions with high concentrations of polar lipids such as phosphatidylethanolamine (PE), sphingomyelin, sphingosphine and lysophosphatidic acid (LPA) in mammalian adipocytes. PMID:27170554

  10. Ancient lipids reveal continuity in culinary practices across the transition to agriculture in Northern Europe

    PubMed Central

    Craig, Oliver E.; Steele, Val J.; Fischer, Anders; Hartz, Sönke; Andersen, Søren H.; Donohoe, Paul; Glykou, Aikaterini; Saul, Hayley; Jones, D. Martin; Koch, Eva; Heron, Carl P.

    2011-01-01

    Farming transformed societies globally. Yet, despite more than a century of research, there is little consensus on the speed or completeness of this fundamental change and, consequently, on its principal drivers. For Northern Europe, the debate has often centered on the rich archaeological record of the Western Baltic, but even here it is unclear how quickly or completely people abandoned wild terrestrial and marine resources after the introduction of domesticated plants and animals at ∼4000 calibrated years B.C. Ceramic containers are found ubiquitously on these sites and contain remarkably well-preserved lipids derived from the original use of the vessel. Reconstructing culinary practices from this ceramic record can contribute to longstanding debates concerning the origins of farming. Here we present data on the molecular and isotopic characteristics of lipids extracted from 133 ceramic vessels and 100 carbonized surface residues dating to immediately before and after the first evidence of domesticated animals and plants in the Western Baltic. The presence of specific lipid biomarkers, notably ω-(o-alkylphenyl)alkanoic acids, and the isotopic composition of individual n-alkanoic acids clearly show that a significant proportion (∼20%) of ceramic vessels with lipids preserved continued to be used for processing marine and freshwater resources across the transition to agriculture in this region. Although changes in pottery use are immediately evident, our data challenge the popular notions that economies were completely transformed with the arrival of farming and that Neolithic pottery was exclusively associated with produce from domesticated animals and plants. PMID:22025697

  11. Ancient lipids reveal continuity in culinary practices across the transition to agriculture in Northern Europe.

    PubMed

    Craig, Oliver E; Steele, Val J; Fischer, Anders; Hartz, Sönke; Andersen, Søren H; Donohoe, Paul; Glykou, Aikaterini; Saul, Hayley; Jones, D Martin; Koch, Eva; Heron, Carl P

    2011-11-01

    Farming transformed societies globally. Yet, despite more than a century of research, there is little consensus on the speed or completeness of this fundamental change and, consequently, on its principal drivers. For Northern Europe, the debate has often centered on the rich archaeological record of the Western Baltic, but even here it is unclear how quickly or completely people abandoned wild terrestrial and marine resources after the introduction of domesticated plants and animals at ∼4000 calibrated years B.C. Ceramic containers are found ubiquitously on these sites and contain remarkably well-preserved lipids derived from the original use of the vessel. Reconstructing culinary practices from this ceramic record can contribute to longstanding debates concerning the origins of farming. Here we present data on the molecular and isotopic characteristics of lipids extracted from 133 ceramic vessels and 100 carbonized surface residues dating to immediately before and after the first evidence of domesticated animals and plants in the Western Baltic. The presence of specific lipid biomarkers, notably ω-(o-alkylphenyl)alkanoic acids, and the isotopic composition of individual n-alkanoic acids clearly show that a significant proportion (∼20%) of ceramic vessels with lipids preserved continued to be used for processing marine and freshwater resources across the transition to agriculture in this region. Although changes in pottery use are immediately evident, our data challenge the popular notions that economies were completely transformed with the arrival of farming and that Neolithic pottery was exclusively associated with produce from domesticated animals and plants. PMID:22025697

  12. Mechanistic insights into EGFR membrane clustering revealed by super-resolution imaging

    NASA Astrophysics Data System (ADS)

    Gao, Jing; Wang, Ye; Cai, Mingjun; Pan, Yangang; Xu, Haijiao; Jiang, Junguang; Ji, Hongbin; Wang, Hongda

    2015-01-01

    The clustering of membrane receptors such as EGFR is critical for various biological processes, for example cell signaling and tumorigenesis. However, the mechanism involved remains poorly understood. Here, we used a super resolution imaging technique, which has shattered the longstanding resolution barrier of light diffraction, to investigate the distribution of membrane EGFR on apical or basal surfaces of COS-7 cells and on the surface of suspended COS-7 cells. Our data show that more and larger EGFR clusters are detected on the apical surface in comparison with those on the basal surface and this difference is not affected by the EGFR activation state, whereas suspended COS-7 cells exhibit a moderate clustering state and a homogeneous distribution pattern, indicating that the external environment surrounding the cell membrane is the decisive factor in the EGFR clustering pattern. A dual-color dSTORM image reveals the significant colocalization of EGFR and lipid rafts; interestingly MβCD treatment leads to a dramatic decrease of the amount and size of EGFR clusters on both apical and basal surfaces, highlighting a key role of lipid rafts in EGFR cluster formation. Altogether, our results illustrate the distribution pattern of EGFR in polarized cells and uncover the essential role of lipid rafts in EGFR cluster maintenance.The clustering of membrane receptors such as EGFR is critical for various biological processes, for example cell signaling and tumorigenesis. However, the mechanism involved remains poorly understood. Here, we used a super resolution imaging technique, which has shattered the longstanding resolution barrier of light diffraction, to investigate the distribution of membrane EGFR on apical or basal surfaces of COS-7 cells and on the surface of suspended COS-7 cells. Our data show that more and larger EGFR clusters are detected on the apical surface in comparison with those on the basal surface and this difference is not affected by the EGFR

  13. Lipidomic profiling of Saccharomyces cerevisiae and Zygosaccharomyces bailii reveals critical changes in lipid composition in response to acetic acid stress.

    PubMed

    Lindberg, Lina; Santos, Aline Xs; Riezman, Howard; Olsson, Lisbeth; Bettiga, Maurizio

    2013-01-01

    When using microorganisms as cell factories in the production of bio-based fuels or chemicals from lignocellulosic hydrolysate, inhibitory concentrations of acetic acid, released from the biomass, reduce the production rate. The undissociated form of acetic acid enters the cell by passive diffusion across the lipid bilayer, mediating toxic effects inside the cell. In order to elucidate a possible link between lipid composition and acetic acid stress, the present study presents detailed lipidomic profiling of the major lipid species found in the plasma membrane, including glycerophospholipids, sphingolipids and sterols, in Saccharomyces cerevisiae (CEN.PK 113_7D) and Zygosaccharomyces bailii (CBS7555) cultured with acetic acid. Detailed physiological characterization of the response of the two yeasts to acetic acid has also been performed in aerobic batch cultivations using bioreactors. Physiological characterization revealed, as expected, that Z. bailii is more tolerant to acetic acid than S. cerevisiae. Z. bailii grew at acetic acid concentrations above 24 g L(-1), while limited growth of S. cerevisiae was observed after 11 h when cultured with only 12 g L(-1) acetic acid. Detailed lipidomic profiling using electrospray ionization, multiple-reaction-monitoring mass spectrometry (ESI-MRM-MS) showed remarkable changes in the glycerophospholipid composition of Z. bailii, including an increase in saturated glycerophospholipids and considerable increases in complex sphingolipids in both S. cerevisiae (IPC 6.2×, MIPC 9.1×, M(IP)2C 2.2×) and Z. bailii (IPC 4.9×, MIPC 2.7×, M(IP)2C 2.7×), when cultured with acetic acid. In addition, the basal level of complex sphingolipids was significantly higher in Z. bailii than in S. cerevisiae, further emphasizing the proposed link between lipid saturation, high sphingolipid levels and acetic acid tolerance. The results also suggest that acetic acid tolerance is associated with the ability of a given strain to generate large

  14. Equilibrium microphase separation in the two-leaflet model of lipid membranes

    NASA Astrophysics Data System (ADS)

    Reigada, Ramon; Mikhailov, Alexander S.

    2016-01-01

    Because of the coupling between local lipid composition and the thickness of the membrane, microphase separation in two-component lipid membranes can take place; such effects may underlie the formation of equilibrium nanoscale rafts. Using a kinetic description, this phenomenon is analytically and numerically investigated. The phase diagram is constructed through the stability analysis for linearized kinetic equations, and conditions for microphase separation are discussed. Simulations of the full kinetic model reveal the development of equilibrium membrane nanostructures with various morphologies from the initial uniform state.

  15. Metabolomics Reveals Altered Lipid Metabolism in a Mouse Model of Endometriosis.

    PubMed

    Dutta, Mainak; Anitha, Mallappa; Smith, Philip B; Chiaro, Christopher R; Maan, Meenu; Chaudhury, Koel; Patterson, Andrew D

    2016-08-01

    Endometriosis is a common chronic estrogen-dependent gynecological disease affecting 10% of women in their reproductive age. It is characterized by proliferation of functional endometrial glands and stroma outside the uterine cavity. In the present study, we used mass spectrometry-based lipidomics to investigate the alterations in serum lipid profiles of mice induced with endometriosis. We identified several dysregulated lipids such as phosphatidylcholines, sphingomyelins, phosphatidylethanolamines, and triglycerides and show that triglycerides may be due to a general inflammatory condition in the peritoneum. We also show that in addition to phosphatidylcholine alteration, there is also an effect in the ratio of phosphatidylcholine/phosphatidylethanolamine in serum of mice induced with the disease and that this change may be due to increased expression of the phosphatidylethanolamine N-methyltransferase gene. The study provides new insight into the etiology of endometriosis. PMID:27246581

  16. Electron tomography reveals the fibril structure and lipid interactions in amyloid deposits.

    PubMed

    Kollmer, Marius; Meinhardt, Katrin; Haupt, Christian; Liberta, Falk; Wulff, Melanie; Linder, Julia; Handl, Lisa; Heinrich, Liesa; Loos, Cornelia; Schmidt, Matthias; Syrovets, Tatiana; Simmet, Thomas; Westermark, Per; Westermark, Gunilla T; Horn, Uwe; Schmidt, Volker; Walther, Paul; Fändrich, Marcus

    2016-05-17

    Electron tomography is an increasingly powerful method to study the detailed architecture of macromolecular complexes or cellular structures. Applied to amyloid deposits formed in a cell culture model of systemic amyloid A amyloidosis, we could determine the structural morphology of the fibrils directly in the deposit. The deposited fibrils are arranged in different networks, and depending on the relative fibril orientation, we can distinguish between fibril meshworks, fibril bundles, and amyloid stars. These networks are frequently infiltrated by vesicular lipid inclusions that may originate from the death of the amyloid-forming cells. Our data support the role of nonfibril components for constructing fibril deposits and provide structural views of different types of lipid-fibril interactions. PMID:27140609

  17. Metabolomics analysis reveals 6‐benzylaminopurine as a stimulator for improving lipid and DHA accumulation of Aurantiochytriumsp.

    PubMed Central

    Yu, Xin‐Jun; Sun, Jie; Zheng, Jian‐Yong; Sun, Ya‐Qi

    2016-01-01

    Abstract BACKGROUND Phytohormones are chemical messengers that have a positive effect on biodiesel production of microalgae at low concentrations. However, the effect of phytohormone 6‐benzylaminopurine on lipid and docosahexaenoic acid (DHA) production in marine DHA‐producer Aurantiochytrium has never been reported. In this study, a GC‐MS‐based metabolomics method combined with a multivariate analysis is applied to reveal the metabolic mechanism of 6‐benzylaminopurine enhancing production of lipid and DHA in Aurantiochytrium sp.YLH70. RESULTS In total, 71 metabolites were identified by GC‐MS. The PCA model revealed that 76.9% of metabolite variation was related to 6‐benzylaminopurine treatment, and overall metabolomics profiles between the 6‐benzylaminopurine and control groups were clearly discriminated. Forty‐six metabolites identified by the PLS‐DA model were responsible for responding to 6‐benzylaminopurine. Metabolic analysis showed that 6‐benzylaminopurine could accelerate the rate of utilization of glucose in Aurantiochytrium sp. YLH70, and the metabolic flux from glycolysis, TCA cycle and mevalonate pathway to fatty acids biosynthesis was promoted. Moreover, the anti‐stress mechanism in Aurantiochytrium sp.YLH70 might be induced by 6‐benzylaminopurine. CONCLUSION Metabolomics is a suitable tool to discover the metabolic mechanism for improving lipid and DHA accumulation in a microorganism. 6‐benzylaminopurine has the potential to stimulate lipid and DHA production of Aurantiochytrium sp.YLH70 for industrial purposes. © 2015 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. PMID:27065509

  18. RAFT polymers for protein recognition

    PubMed Central

    Tominey, Alan F; Liese, Julia; Wei, Sun; Kowski, Klaus

    2010-01-01

    Summary A new family of linear polymers with pronounced affinity for arginine- and lysine-rich proteins has been created. To this end, N-isopropylacrylamide (NIPAM) was copolymerized in water with a binding monomer and a hydrophobic comonomer using a living radical polymerization (RAFT). The resulting copolymers were water-soluble and displayed narrow polydispersities. They formed tight complexes with basic proteins depending on the nature and amount of the binding monomer as well as on the choice of the added hydrophobic comonomer. PMID:20703378

  19. 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.…

  20. Stabilization of concentration fluctuations in mixed membranes by hybrid lipids

    NASA Astrophysics Data System (ADS)

    Palmieri, Benoit; Safran, Samuel

    2012-02-01

    Finite-size domains have been observed at the surface of cells. These lipids ``rafts'' are stable nanodomains enriched in saturated lipids and cholesterol. While line tension favors macrodomains, one explanation for raft stabilization suggests that the membrane composition is tuned close to a spinodal temperature. From this point of view, rafts are long-lived concentration fluctuations in the mixed phase. We propose a ternary mixture model for the cell membrane that includes hybrid lipids which have one saturated and one unsaturated hydrocarbon chain. Finite amount of hybrid lipids reduces the packing incompatibility at the saturated/unsaturated lipid interface and stabilizes the concentration fluctuations. Hybrid-Hybrid interactions are included in the model and further increase the life-time of the rafts and decrease their length-scales. Moreover, the hybrid has extra orientational degrees of freedom that may lead to modulated phases.

  1. Reconstruction of the lipid metabolism for the microalga Monoraphidium neglectum from its genome sequence reveals characteristics suitable for biofuel production

    PubMed Central

    2013-01-01

    Background Microalgae are gaining importance as sustainable production hosts in the fields of biotechnology and bioenergy. A robust biomass accumulating strain of the genus Monoraphidium (SAG 48.87) was investigated in this work as a potential feedstock for biofuel production. The genome was sequenced, annotated, and key enzymes for triacylglycerol formation were elucidated. Results Monoraphidium neglectum was identified as an oleaginous species with favourable growth characteristics as well as a high potential for crude oil production, based on neutral lipid contents of approximately 21% (dry weight) under nitrogen starvation, composed of predominantly C18:1 and C16:0 fatty acids. Further characterization revealed growth in a relatively wide pH range and salt concentrations of up to 1.0% NaCl, in which the cells exhibited larger structures. This first full genome sequencing of a member of the Selenastraceae revealed a diploid, approximately 68 Mbp genome with a G + C content of 64.7%. The circular chloroplast genome was assembled to a 135,362 bp single contig, containing 67 protein-coding genes. The assembly of the mitochondrial genome resulted in two contigs with an approximate total size of 94 kb, the largest known mitochondrial genome within algae. 16,761 protein-coding genes were assigned to the nuclear genome. Comparison of gene sets with respect to functional categories revealed a higher gene number assigned to the category “carbohydrate metabolic process” and in “fatty acid biosynthetic process” in M. neglectum when compared to Chlamydomonas reinhardtii and Nannochloropsis gaditana, indicating a higher metabolic diversity for applications in carbohydrate conversions of biotechnological relevance. Conclusions The genome of M. neglectum, as well as the metabolic reconstruction of crucial lipid pathways, provides new insights into the diversity of the lipid metabolism in microalgae. The results of this work provide a platform to encourage the

  2. Initial steps of Shigella infection depend on the cholesterol/sphingolipid raft-mediated CD44-IpaB interaction.

    PubMed

    Lafont, Frank; Tran Van Nhieu, Guy; Hanada, Kentaro; Sansonetti, Philippe; van der Goot, F Gisou

    2002-09-01

    Shigellosis is an acute inflammatory bowel disease caused by the enteroinvasive bacterium SHIGELLA: Upon host cell-Shigella interaction, major host cell signalling responses are activated. Deciphering the initial molecular events is crucial to understanding the infectious process. We identified a molecular complex involving proteins of both the host, CD44 the hyaluronan receptor, and Shigella, the invasin IpaB, which partitions during infection within specialized membrane microdomains enriched in cholesterol and sphingolipids, called rafts. We also document accumulation of cholesterol and raft-associated proteins at Shigella entry foci. Moreover, we report that Shigella entry is impaired after cholesterol depletion using methyl-beta-cyclodextrin. Finally, we find that Shigella is less invasive in sphingosid-based lipid-deficient cell lines, demonstrating the involvement of sphingolipids. Our results show that rafts are implicated in Shigella binding and entry, suggesting that raft-associated molecular machineries are engaged in mediating the cell signalling response required for the invasion process. PMID:12198147

  3. Lipid Profiling and Transcriptomic Analysis Reveals a Functional Interplay between Estradiol and Growth Hormone in Liver

    PubMed Central

    Fernández-Pérez, Leandro; Santana-Farré, Ruymán; de Mirecki-Garrido, Mercedes; García, Irma; Guerra, Borja; Mateo-Díaz, Carlos; Iglesias-Gato, Diego; Díaz-Chico, Juan Carlos; Flores-Morales, Amilcar; Díaz, Mario

    2014-01-01

    17β-estradiol (E2) may interfere with endocrine, metabolic, and gender-differentiated functions in liver in both females and males. Indirect mechanisms play a crucial role because of the E2 influence on the pituitary GH secretion and the GHR-JAK2-STAT5 signaling pathway in the target tissues. E2, through its interaction with the estrogen receptor, exerts direct effects on liver. Hypothyroidism also affects endocrine and metabolic functions of the liver, rendering a metabolic phenotype with features that mimic deficiencies in E2 or GH. In this work, we combined the lipid and transcriptomic analysis to obtain comprehensive information on the molecular mechanisms of E2 effects, alone and in combination with GH, to regulate liver functions in males. We used the adult hypothyroid-orchidectomized rat model to minimize the influence of internal hormones on E2 treatment and to explore its role in male-differentiated functions. E2 influenced genes involved in metabolism of lipids and endo-xenobiotics, and the GH-regulated endocrine, metabolic, immune, and male-specific responses. E2 induced a female-pattern of gene expression and inhibited GH-regulated STAT5b targeted genes. E2 did not prevent the inhibitory effects of GH on urea and amino acid metabolism-related genes. The combination of E2 and GH decreased transcriptional immune responses. E2 decreased the hepatic content of saturated fatty acids and induced a transcriptional program that seems to be mediated by the activation of PPARα. In contrast, GH inhibited fatty acid oxidation. Both E2 and GH replacements reduced hepatic CHO levels and increased the formation of cholesterol esters and triacylglycerols. Notably, the hepatic lipid profiles were endowed with singular fingerprints that may be used to segregate the effects of different hormonal replacements. In summary, we provide in vivo evidence that E2 has a significant impact on lipid content and transcriptome in male liver and that E2 exerts a marked influence on

  4. Metabonomics Reveals Drastic Changes in Anti-Inflammatory/Pro-Resolving Polyunsaturated Fatty Acids-Derived Lipid Mediators in Leprosy Disease

    PubMed Central

    Amaral, Julio J.; Antunes, Luis Caetano M.; de Macedo, Cristiana S.; Mattos, Katherine A.; Han, Jun; Pan, Jingxi; Candéa, André L. P.; Henriques, Maria das Graças M. O.; Ribeiro-Alves, Marcelo; Borchers, Christoph H.; Sarno, Euzenir N.; Bozza, Patrícia T.; Finlay, B. Brett; Pessolani, Maria Cristina V.

    2013-01-01

    Despite considerable efforts over the last decades, our understanding of leprosy pathogenesis remains limited. The complex interplay between pathogens and hosts has profound effects on host metabolism. To explore the metabolic perturbations associated with leprosy, we analyzed the serum metabolome of leprosy patients. Samples collected from lepromatous and tuberculoid patients before and immediately after the conclusion of multidrug therapy (MDT) were subjected to high-throughput metabolic profiling. Our results show marked metabolic alterations during leprosy that subside at the conclusion of MDT. Pathways showing the highest modulation were related to polyunsaturated fatty acid (PUFA) metabolism, with emphasis on anti-inflammatory, pro-resolving omega-3 fatty acids. These results were confirmed by eicosanoid measurements through enzyme-linked immunoassays. Corroborating the repertoire of metabolites altered in sera, metabonomic analysis of skin specimens revealed alterations in the levels of lipids derived from lipase activity, including PUFAs, suggesting a high lipid turnover in highly-infected lesions. Our data suggest that omega-6 and omega-3, PUFA-derived, pro-resolving lipid mediators contribute to reduced tissue damage irrespectively of pathogen burden during leprosy disease. Our results demonstrate the utility of a comprehensive metabonomic approach for identifying potential contributors to disease pathology that may facilitate the development of more targeted treatments for leprosy and other inflammatory diseases. PMID:23967366

  5. Driving force for crystallization of anionic lipid membranes revealed by atomistic simulations.

    PubMed

    Qiao, Bao Fu; Olvera de la Cruz, Monica

    2013-05-01

    Crystalline vesicles are promising nanomaterials due to their mechanical stability in various environments. To control their fabrication, it is essential to understand the effects of different experimental conditions on crystallization. Here we perform atomistic molecular dynamics simulations of anionic lipid membranes of 1,2-dilauroyl-sn-glycero-3-phosphol-L-serine. In the presence of Na(+) monovalent counterions, we access the phase transition from the liquid-like disordered liquid-crystalline phase to the ordered gel phase by lowering the temperature of the system. The phase transition is conclusively evidenced by the scattering structure factor. Quantitative calculations show that the enhancement of the intertail van der Waals interaction (about -6 k(B)T) plays a dominant role in driving the phase transition rather than the increase in the cohesive interaction (-0.5 k(B)T) between lipids and counterions. Meanwhile, in the presence of multivalent counterions of Zn(2+) or La(3+) the gel phase is found throughout the temperature range investigated. Moreover, the van der Waals interaction per hydrocarbon group is ∼20% stronger in the gel phase (∼ -1.8 k(B)T regardless of the counterions) than in the liquid-crystalline phase (-1.5 k(B)T). PMID:23565965

  6. Metabolomics of reef benthic interactions reveals a bioactive lipid involved in coral defence.

    PubMed

    Quinn, Robert A; Vermeij, Mark J A; Hartmann, Aaron C; Galtier d'Auriac, Ines; Benler, Sean; Haas, Andreas; Quistad, Steven D; Lim, Yan Wei; Little, Mark; Sandin, Stuart; Smith, Jennifer E; Dorrestein, Pieter C; Rohwer, Forest

    2016-04-27

    Holobionts are assemblages of microbial symbionts and their macrobial host. As extant representatives of some of the oldest macro-organisms, corals and algae are important for understanding how holobionts develop and interact with one another. Using untargeted metabolomics, we show that non-self interactions altered the coral metabolome more than self-interactions (i.e. different or same genus, respectively). Platelet activating factor (PAF) and Lyso-PAF, central inflammatory modulators in mammals, were major lipid components of the coral holobionts. When corals were damaged during competitive interactions with algae, PAF increased along with expression of the gene encoding Lyso-PAF acetyltransferase; the protein responsible for converting Lyso-PAF to PAF. This shows that self and non-self recognition among some of the oldest extant holobionts involve bioactive lipids identical to those in highly derived taxa like humans. This further strengthens the hypothesis that major players of the immune response evolved during the pre-Cambrian. PMID:27122568

  7. Metabolomics of reef benthic interactions reveals a bioactive lipid involved in coral defence

    PubMed Central

    Vermeij, Mark J. A.; Hartmann, Aaron C.; Galtier d'Auriac, Ines; Benler, Sean; Haas, Andreas; Quistad, Steven D.; Lim, Yan Wei; Little, Mark; Sandin, Stuart; Smith, Jennifer E.; Dorrestein, Pieter C.; Rohwer, Forest

    2016-01-01

    Holobionts are assemblages of microbial symbionts and their macrobial host. As extant representatives of some of the oldest macro-organisms, corals and algae are important for understanding how holobionts develop and interact with one another. Using untargeted metabolomics, we show that non-self interactions altered the coral metabolome more than self-interactions (i.e. different or same genus, respectively). Platelet activating factor (PAF) and Lyso-PAF, central inflammatory modulators in mammals, were major lipid components of the coral holobionts. When corals were damaged during competitive interactions with algae, PAF increased along with expression of the gene encoding Lyso-PAF acetyltransferase; the protein responsible for converting Lyso-PAF to PAF. This shows that self and non-self recognition among some of the oldest extant holobionts involve bioactive lipids identical to those in highly derived taxa like humans. This further strengthens the hypothesis that major players of the immune response evolved during the pre-Cambrian. PMID:27122568

  8. Exploration of scalp surface lipids reveals squalene peroxide as a potential actor in dandruff condition.

    PubMed

    Jourdain, Roland; Moga, Alain; Vingler, Philippe; El Rawadi, Charles; Pouradier, Florence; Souverain, Luc; Bastien, Philippe; Amalric, Nicolas; Breton, Lionel

    2016-04-01

    Dandruff is a common but complex disorder with three major contributing factors: (1) individual predisposition, (2) scalp sebum and (3) Malassezia yeast colonization. To obtain further insights into the role of sebum in dandruff biogenesis, we analyzed scalp lipid species in a cohort of ten dandruff-free (control) and ten dandruff-afflicted volunteers by gas chromatography coupled to mass spectrometry. Lipid peroxidation levels and biochemical markers of oxidative stress were also assessed. Squalene, a major sebum component, was significantly more peroxidized in dandruff-affected scalps, resulting in significantly higher ratios of squalene monohydroperoxide (SQOOH)/squalene. This was observed when comparing dandruff-affected zones of dandruff subjects to both their non-affected zones and control subjects. In addition, other biomarkers such as malondialdehyde indicated that oxidative stress levels were raised on dandruff scalps. Surprisingly, differences regarding either free or bound fatty acids were fairly rare and minor. Certain novel findings, especially squalene peroxidation levels, were then confirmed in a validation cohort of 24 dandruff-affected subjects, by comparing dandruff-affected and non-dandruff zones from the same individuals. As SQOOH can induce both keratinocyte inflammatory responses and hyperproliferation in vitro, we hypothesized that increased SQOOH could be considered as a new etiological dandruff factor via its ability to impair scalp barrier function. Our results also indicated that Malassezia could be a major source of squalene peroxidation on the scalp. PMID:26842231

  9. Lateral organization of complex lipid mixtures from multiscale modeling

    NASA Astrophysics Data System (ADS)

    Tumaneng, Paul W.; Pandit, Sagar A.; Zhao, Guijun; Scott, H. L.

    2010-02-01

    The organizational properties of complex lipid mixtures can give rise to functionally important structures in cell membranes. In model membranes, ternary lipid-cholesterol (CHOL) mixtures are often used as representative systems to investigate the formation and stabilization of localized structural domains ("rafts"). In this work, we describe a self-consistent mean-field model that builds on molecular dynamics simulations to incorporate multiple lipid components and to investigate the lateral organization of such mixtures. The model predictions reveal regions of bimodal order on ternary plots that are in good agreement with experiment. Specifically, we have applied the model to ternary mixtures composed of dioleoylphosphatidylcholine:18:0 sphingomyelin:CHOL. This work provides insight into the specific intermolecular interactions that drive the formation of localized domains in these mixtures. The model makes use of molecular dynamics simulations to extract interaction parameters and to provide chain configuration order parameter libraries.

  10. Removal of silica from Raft River geothermal water

    SciTech Connect

    Suciu, D.F.; Miller, R.L.

    1980-06-01

    Lack of sufficient quantities of clean surface or near-surface water at Raft River for cooling purposes dictates that cooled geothermal fluid, effluent from the Raft River 5 MW(e) Pilot Power Plant, must also be used as condenser coolant. Prior testing revealed that a water-treatment system would be required to reduce silica and calcium concentrations of the cooling fluid. The water-treatment system specified was to use dolomitic lime for both pH adjustment and source of magnesium. The dolomitic lime treatment was investigated and found to be inadequate. Subsequent testing was done to find chemical systems that would adequately reduce silica concentrations. Three magnesium and two iron compounds were found which reduced silica to acceptable concentration levels. They are magnesium bicarbonate, magnesium chloride, magnesium sulfate, iron sulfate, and iron chloride. Magnesium oxide, using a two-stage countercurrent process, will also reduce silica to adequate levels.

  11. HDL surface lipids mediate CETP binding as revealed by electron microscopy and molecular dynamics simulation

    PubMed Central

    Zhang, Meng; Charles, River; Tong, Huimin; Zhang, Lei; Patel, Mili; Wang, Francis; Rames, Matthew J.; Ren, Amy; Rye, Kerry-Anne; Qiu, Xiayang; Johns, Douglas G.; Charles, M. Arthur; Ren, Gang

    2015-01-01

    Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesterol esters (CE) from atheroprotective high-density lipoproteins (HDL) to atherogenic low-density lipoproteins (LDL). CETP inhibition has been regarded as a promising strategy for increasing HDL levels and subsequently reducing the risk of cardiovascular diseases (CVD). Although the crystal structure of CETP is known, little is known regarding how CETP binds to HDL. Here, we investigated how various HDL-like particles interact with CETP by electron microscopy and molecular dynamics simulations. Results showed that CETP binds to HDL via hydrophobic interactions rather than protein-protein interactions. The HDL surface lipid curvature generates a hydrophobic environment, leading to CETP hydrophobic distal end interaction. This interaction is independent of other HDL components, such as apolipoproteins, cholesteryl esters and triglycerides. Thus, disrupting these hydrophobic interactions could be a new therapeutic strategy for attenuating the interaction of CETP with HDL. PMID:25737239

  12. HDL surface lipids mediate CETP binding as revealed by electron microscopy and molecular dynamics simulation

    DOE PAGESBeta

    Zhang, Meng; Charles, River; Tong, Huimin; Zhang, Lei; Patel, Mili; Wang, Francis; Rames, Matthew J.; Ren, Amy; Rye, Kerry-Anne; Qiu, Xiayang; et al

    2015-03-04

    Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesterol esters (CE) from atheroprotective high-density lipoproteins (HDL) to atherogenic low-density lipoproteins (LDL). CETP inhibition has been regarded as a promising strategy for increasing HDL levels and subsequently reducing the risk of cardiovascular diseases (CVD). Although the crystal structure of CETP is known, little is known regarding how CETP binds to HDL. Here, we investigated how various HDL-like particles interact with CETP by electron microscopy and molecular dynamics simulations. Results showed that CETP binds to HDL via hydrophobic interactions rather than protein-protein interactions. The HDL surface lipid curvature generates a hydrophobicmore » environment, leading to CETP hydrophobic distal end interaction. This interaction is independent of other HDL components, such as apolipoproteins, cholesteryl esters and triglycerides. Thus, disrupting these hydrophobic interactions could be a new therapeutic strategy for attenuating the interaction of CETP with HDL.« less

  13. HDL surface lipids mediate CETP binding as revealed by electron microscopy and molecular dynamics simulation

    SciTech Connect

    Zhang, Meng; Charles, River; Tong, Huimin; Zhang, Lei; Patel, Mili; Wang, Francis; Rames, Matthew J.; Ren, Amy; Rye, Kerry-Anne; Qiu, Xiayang; Johns, Douglas G.; Charles, M. Arthur; Ren, Gang

    2015-03-04

    Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesterol esters (CE) from atheroprotective high-density lipoproteins (HDL) to atherogenic low-density lipoproteins (LDL). CETP inhibition has been regarded as a promising strategy for increasing HDL levels and subsequently reducing the risk of cardiovascular diseases (CVD). Although the crystal structure of CETP is known, little is known regarding how CETP binds to HDL. Here, we investigated how various HDL-like particles interact with CETP by electron microscopy and molecular dynamics simulations. Results showed that CETP binds to HDL via hydrophobic interactions rather than protein-protein interactions. The HDL surface lipid curvature generates a hydrophobic environment, leading to CETP hydrophobic distal end interaction. This interaction is independent of other HDL components, such as apolipoproteins, cholesteryl esters and triglycerides. Thus, disrupting these hydrophobic interactions could be a new therapeutic strategy for attenuating the interaction of CETP with HDL.

  14. HDL surface lipids mediate CETP binding as revealed by electron microscopy and molecular dynamics simulation

    NASA Astrophysics Data System (ADS)

    Zhang, Meng; Charles, River; Tong, Huimin; Zhang, Lei; Patel, Mili; Wang, Francis; Rames, Matthew J.; Ren, Amy; Rye, Kerry-Anne; Qiu, Xiayang; Johns, Douglas G.; Charles, M. Arthur; Ren, Gang

    2015-03-01

    Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesterol esters (CE) from atheroprotective high-density lipoproteins (HDL) to atherogenic low-density lipoproteins (LDL). CETP inhibition has been regarded as a promising strategy for increasing HDL levels and subsequently reducing the risk of cardiovascular diseases (CVD). Although the crystal structure of CETP is known, little is known regarding how CETP binds to HDL. Here, we investigated how various HDL-like particles interact with CETP by electron microscopy and molecular dynamics simulations. Results showed that CETP binds to HDL via hydrophobic interactions rather than protein-protein interactions. The HDL surface lipid curvature generates a hydrophobic environment, leading to CETP hydrophobic distal end interaction. This interaction is independent of other HDL components, such as apolipoproteins, cholesteryl esters and triglycerides. Thus, disrupting these hydrophobic interactions could be a new therapeutic strategy for attenuating the interaction of CETP with HDL.

  15. A lipidomics study reveals hepatic lipid signatures associating with deficiency of the LDL receptor in a rat model

    PubMed Central

    Quan, Chao; Hu, Chunxiu; Xie, Bingxian; Du, Yinan; Chen, Liang; Yang, Wei; Yang, Liu; Chen, Qiaoli; Shen, Bin; Hu, Bian; Zheng, Zhihong; Zhu, Haibo; Huang, Xingxu; Xu, Guowang; Chen, Shuai

    2016-01-01

    ABSTRACT The low-density lipoprotein receptor (LDLR) plays a critical role in the liver for the clearance of plasma low-density lipoprotein (LDL). Its deficiency causes hypercholesterolemia in many models. To facilitate the usage of rats as animal models for the discovery of cholesterol-lowering drugs, we took a genetic approach to delete the LDLR in rats aiming to increase plasma LDL cholesterol (LDL-C). An LDLR knockout rat was generated via zinc-finger nuclease technology, which harbors a 19-basepair deletion in the seventh exon of the ldlr gene. As expected, deletion of the LDLR elevated total cholesterol and total triglyceride in the plasma, and caused a tenfold increase of plasma LDL-C and a fourfold increase of plasma very low-density lipoprotein (VLDL-C). A lipidomics analysis revealed that deletion of the LDLR affected hepatic lipid metabolism, particularly lysophosphatidylcholines, free fatty acids and sphingolipids in the liver. Cholesterol ester (CE) 20:4 also displayed a significant increase in the LDLR knockout rats. Taken together, the LDLR knockout rat offers a new model of hypercholesterolemia, and the lipidomics analysis reveals hepatic lipid signatures associating with deficiency of the LDL receptor. PMID:27378433

  16. A lipidomics study reveals hepatic lipid signatures associating with deficiency of the LDL receptor in a rat model.

    PubMed

    Wang, Hong Yu; Quan, Chao; Hu, Chunxiu; Xie, Bingxian; Du, Yinan; Chen, Liang; Yang, Wei; Yang, Liu; Chen, Qiaoli; Shen, Bin; Hu, Bian; Zheng, Zhihong; Zhu, Haibo; Huang, Xingxu; Xu, Guowang; Chen, Shuai

    2016-01-01

    The low-density lipoprotein receptor (LDLR) plays a critical role in the liver for the clearance of plasma low-density lipoprotein (LDL). Its deficiency causes hypercholesterolemia in many models. To facilitate the usage of rats as animal models for the discovery of cholesterol-lowering drugs, we took a genetic approach to delete the LDLR in rats aiming to increase plasma LDL cholesterol (LDL-C). An LDLR knockout rat was generated via zinc-finger nuclease technology, which harbors a 19-basepair deletion in the seventh exon of the ldlr gene. As expected, deletion of the LDLR elevated total cholesterol and total triglyceride in the plasma, and caused a tenfold increase of plasma LDL-C and a fourfold increase of plasma very low-density lipoprotein (VLDL-C). A lipidomics analysis revealed that deletion of the LDLR affected hepatic lipid metabolism, particularly lysophosphatidylcholines, free fatty acids and sphingolipids in the liver. Cholesterol ester (CE) 20:4 also displayed a significant increase in the LDLR knockout rats. Taken together, the LDLR knockout rat offers a new model of hypercholesterolemia, and the lipidomics analysis reveals hepatic lipid signatures associating with deficiency of the LDL receptor. PMID:27378433

  17. Femtosecond Hydrogen Bond Dynamics of Bulk-like and Bound Water at Positively and Negatively Charged Lipid Interfaces Revealed by 2D HD-VSFG Spectroscopy.

    PubMed

    Singh, Prashant Chandra; Inoue, Ken-Ichi; Nihonyanagi, Satoshi; Yamaguchi, Shoichi; Tahara, Tahei

    2016-08-26

    Interfacial water in the vicinity of lipids plays an important role in many biological processes, such as drug delivery, ion transportation, and lipid fusion. Hence, molecular-level elucidation of the properties of water at lipid interfaces is of the utmost importance. We report the two-dimensional heterodyne-detected vibrational sum frequency generation (2D HD-VSFG) study of the OH stretch of HOD at charged lipid interfaces, which shows that the hydrogen bond dynamics of interfacial water differ drastically, depending on the lipids. The data indicate that the spectral diffusion of the OH stretch at a positively charged lipid interface is dominated by the ultrafast (<∼100 fs) component, followed by the minor sub-picosecond slow dynamics, while the dynamics at a negatively charged lipid interface exhibit sub-picosecond dynamics almost exclusively, implying that fast hydrogen bond fluctuation is prohibited. These results reveal that the ultrafast hydrogen bond dynamics at the positively charged lipid-water interface are attributable to the bulk-like property of interfacial water, whereas the slow dynamics at the negatively charged lipid interface are due to bound water, which is hydrogen-bonded to the hydrophilic head group. PMID:27482947

  18. Metabolomics approach reveals effects of antihypertensives and lipid-lowering drugs on the human metabolism.

    PubMed

    Altmaier, Elisabeth; Fobo, Gisela; Heier, Margit; Thorand, Barbara; Meisinger, Christine; Römisch-Margl, Werner; Waldenberger, Melanie; Gieger, Christian; Illig, Thomas; Adamski, Jerzy; Suhre, Karsten; Kastenmüller, Gabi

    2014-05-01

    The mechanism of antihypertensive and lipid-lowering drugs on the human organism is still not fully understood. New insights on the drugs' action can be provided by a metabolomics-driven approach, which offers a detailed view of the physiological state of an organism. Here, we report a metabolome-wide association study with 295 metabolites in human serum from 1,762 participants of the KORA F4 (Cooperative Health Research in the Region of Augsburg) study population. Our intent was to find variations of metabolite concentrations related to the intake of various drug classes and--based on the associations found--to generate new hypotheses about on-target as well as off-target effects of these drugs. In total, we found 41 significant associations for the drug classes investigated: For beta-blockers (11 associations), angiotensin-converting enzyme (ACE) inhibitors (four assoc.), diuretics (seven assoc.), statins (ten assoc.), and fibrates (nine assoc.) the top hits were pyroglutamine, phenylalanylphenylalanine, pseudouridine, 1-arachidonoylglycerophosphocholine, and 2-hydroxyisobutyrate, respectively. For beta-blockers we observed significant associations with metabolite concentrations that are indicative of drug side-effects, such as increased serotonin and decreased free fatty acid levels. Intake of ACE inhibitors and statins associated with metabolites that provide insight into the action of the drug itself on its target, such as an association of ACE inhibitors with des-Arg(9)-bradykinin and aspartylphenylalanine, a substrate and a product of the drug-inhibited ACE. The intake of statins which reduce blood cholesterol levels, resulted in changes in the concentration of metabolites of the biosynthesis as well as of the degradation of cholesterol. Fibrates showed the strongest association with 2-hydroxyisobutyrate which might be a breakdown product of fenofibrate and, thus, a possible marker for the degradation of this drug in the human organism. The analysis of

  19. Squalene synthase, a determinant of Raft-associated cholesterol and modulator of cancer cell proliferation.

    PubMed

    Brusselmans, Koen; Timmermans, Leen; Van de Sande, Tine; Van Veldhoven, Paul P; Guan, Guimin; Shechter, Ishaiahu; Claessens, Frank; Verhoeven, Guido; Swinnen, Johannes V

    2007-06-29

    Several cues for cell proliferation, migration, and survival are transmitted through lipid rafts, membrane microdomains enriched in sphingolipids and cholesterol. Cells obtain cholesterol from the circulation but can also synthesize cholesterol de novo through the mevalonate/isoprenoid pathway. This pathway, however, has several branches and also produces non-sterol isoprenoids. Squalene synthase (SQS) is the enzyme that determines the switch toward sterol biosynthesis. Here we demonstrate that in prostate cancer cells SQS expression is enhanced by androgens, channeling intermediates of the mevalonate/isoprenoid pathway toward cholesterol synthesis. Interestingly, the resulting increase in de novo synthesis of cholesterol mainly affects the cholesterol content of lipid rafts, while leaving non-raft cholesterol levels unaffected. Conversely, RNA interference-mediated SQS inhibition results in a decrease of raft-associated cholesterol. These data show that SQS activity and de novo cholesterol synthesis are determinants of membrane microdomain-associated cholesterol in cancer cells. Remarkably, SQS knock down also attenuates proliferation and induces death of prostate cancer cells. Similar effects are observed when cancer cells are treated with the chemical SQS inhibitor zaragozic acid A. Importantly, although the anti-tumor effect of statins has previously been attributed to inhibition of protein isoprenylation, the present study shows that specific inhibition of the cholesterol biosynthesis branch of the mevalonate/isoprenoid pathway also induces cancer cell death. These findings significantly underscore the importance of de novo cholesterol synthesis for cancer cell biology and suggest that SQS is a potential novel target for antineoplastic intervention. PMID:17483544

  20. Untargeted metabolomic analysis of miltefosine action in Leishmania infantum reveals changes to the internal lipid metabolism☆

    PubMed Central

    Vincent, Isabel M.; Weidt, Stefan; Rivas, Luis; Burgess, Karl; Smith, Terry K.; Ouellette, Marc

    2013-01-01

    There are many theories as to the mode of action of miltefosine against Leishmania including alterations to the membrane lipid content, induction of apoptosis and modulation of macrophage responses. Here we perform untargeted metabolomics to elucidate the metabolic changes involved in miltefosine action. Over 800 metabolites were detected, 10% of which were significantly altered after 3.75 h. Many of the changes related to an increase in alkane fragment and sugar release. Fragment release is synchronised with reactive oxygen species production, but native membrane phospholipids remain intact. Signs of DNA damage were also detected as were changes to the levels of some thiols and polyamines. After 5 h of miltefosine treatment the cells showed depleted levels of most metabolites, indicating that the cells’ outer membrane integrity had become compromised and internal metabolites were escaping upon cell death. In miltefosine resistant cells, the drug was not internalised and the changes to the internal metabolite levels were not seen. In contrast, cells resistant to antimony (SbIII) had similar corresponding alterations to the levels of internal metabolites as wild-type cells. A detailed knowledge of the mode of action of miltefosine will be important to inform the design of combination therapies to combat leishmaniasis, something that the research community should be prioritising in the coming years. PMID:24596665

  1. Size and orientation of the lipid II headgroup as revealed by AFM imaging.

    PubMed

    Ganchev, D N; Hasper, H E; Breukink, E; de Kruijff, B

    2006-05-16

    In this study, we investigated the size and orientation of the bacterial Lipid II (L II) headgroup when the L II molecule is present in liquid-crystalline domains of DOPC in a supported DPPC bilayer. Using atomic force microscopy, we detected that L II causes the appearance of a 1.9 nm thick layer, situated over the DOPC headgroup region. With an increased scanning force, this layer can be penetrated by the AFM tip down to the level of the DOPC bilayer. Using different L II precursor molecules, we demonstrated that the detected layer consists of the headgroups of L II and that the MurNAc-pentapeptide unit of the headgroup is responsible for the measured 1.9 nm height of that layer. Monolayer experiments provided information about the in-plane dimensions of the L II headgroup. On the basis of these results and considerations of the molecular dimensions of L II headgroup constituents, we propose a model for the orientation of the L II headgroup in the membrane. In this model, the pentapeptide of the L II headgroup is rather extended and points away from the bilayer surface, which could be important for biological processes, in which L II is involved. PMID:16681392

  2. Pattern Formation in the Arabidopsis Embryo Revealed by Position-Specific Lipid Transfer Protein Gene Expression.

    PubMed Central

    Vroemen, C. W.; Langeveld, S.; Mayer, U.; Ripper, G.; Jurgens, G.; Van Kammen, A.; De Vries, S. C.

    1996-01-01

    During Arabidopsis embryogenesis, the zygote divides asymmetrically in the future apical-basal axis; however, a radial axis is initiated only within the eight-celled embryo. Mutations in the GNOM, KNOLLE, and KEULE genes affect these processes: gnom zygotes tend to divide symmetrically; knolle embryos lack oriented cell divisions that initiate protoderm formation; and in keule embryos, an outer cell layer is present that consists of abnormally enlarged cells from early development. Pattern formation along the two axes is reflected by the position-specific expression of the Arabidopsis lipid transfer protein (AtLTP1) gene. In wild-type embryos, the AtLTP1 gene is expressed in the protoderm and initially in all protodermal cells; later, AtLTP1 expression is confined to the cotyledons and the upper end of the hypocotyl. Analysis of AtLTP1 expression in gnom, knolle, and keule embryos showed that gnom embryos also can have no or reversed apical-basal polarity, whereas radial polarity is unaffected. knolle embryos initially lack but eventually form a radial pattern, and keule embryos are affected in protoderm cell morphology rather than in the establishment of the radial pattern. PMID:12239400

  3. Raft River geoscience case study: appendixes

    SciTech Connect

    Dolenc, M.R.; Hull, L.C.; Mizell, S.A.; Russell, B.F.; Skiba, P.A.; Strawn, J.A.; Tullis, J.A.

    1981-11-01

    The following are included in these appendices: lithology, x-ray analysis, and cores; well construction data; borehole geophysical logs; chemical analyses from wells at the Raft River geothermal site; and bibliography. (MHR)

  4. Metagenomic and lipid analyses reveal a diel cycle in a hypersaline microbial ecosystem.

    PubMed

    Andrade, Karen; Logemann, Jörn; Heidelberg, Karla B; Emerson, Joanne B; Comolli, Luis R; Hug, Laura A; Probst, Alexander J; Keillar, Angus; Thomas, Brian C; Miller, Christopher S; Allen, Eric E; Moreau, John W; Brocks, Jochen J; Banfield, Jillian F

    2015-12-01

    Marine microbial communities experience daily fluctuations in light and temperature that can have important ramifications for carbon and nutrient cycling. Elucidation of such short time scale community-wide dynamics is hindered by system complexity. Hypersaline aquatic environments have lower species richness than marine environments and can be well-defined spatially, hence they provide a model system for diel cycle analysis. We conducted a 3-day time series experiment in a well-defined pool in hypersaline Lake Tyrrell, Australia. Microbial communities were tracked by combining cultivation-independent lipidomic, metagenomic and microscopy methods. The ratio of total bacterial to archaeal core lipids in the planktonic community increased by up to 58% during daylight hours and decreased by up to 32% overnight. However, total organism abundances remained relatively consistent over 3 days. Metagenomic analysis of the planktonic community composition, resolved at the genome level, showed dominance by Haloquadratum species and six uncultured members of the Halobacteriaceae. The post 0.8 μm filtrate contained six different nanohaloarchaeal types, three of which have not been identified previously, and cryo-transmission electron microscopy imaging confirmed the presence of small cells. Notably, these nano-sized archaea showed a strong diel cycle, with a pronounced increase in relative abundance over the night periods. We detected no eukaryotic algae or other photosynthetic primary producers, suggesting that carbon resources may derive from patchily distributed microbial mats at the sediment-water interface or from surrounding land. Results show the operation of a strong community-level diel cycle, probably driven by interconnected temperature, light abundance, dissolved oxygen concentration and nutrient flux effects. PMID:25918833

  5. Metabolic labelling of membrane microdomains/rafts in Jurkat cells indicates the presence of glycerophospholipids implicated in signal transduction by the CD3 T-cell receptor.

    PubMed Central

    Rouquette-Jazdanian, Alexandre K; Pelassy, Claudette; Breittmayer, Jean-Philippe; Cousin, Jean-Louis; Aussel, Claude

    2002-01-01

    Cell membranes contain sphingolipids and cholesterol, which cluster together in distinct domains called rafts. The outer-membrane leaflet of these peculiar membrane domains contains glycosylphosphatidylinositol-anchored proteins, while the inner leaflet contains proteins implicated in signalling, such as the acylated protein kinase p56(lck) and the palmitoylated adaptator LAT (linker for activation of T-cells). We present here an approach to study the lipid composition of rafts and its change upon T-cell activation. Our method is based on metabolic labelling of Jurkat T-cells with different precursors of glycerophospholipid synthesis, including glycerol and fatty acids with different lengths and degrees of saturation as well as phospholipid polar head groups. The results obtained indicate that lipid rafts isolated by the use of sucrose density-gradient centrifugation after Triton X-100 extraction in the cold, besides sphingolipids and cholesterol, contain unambiguously all classes of glycerophospholipids: phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine. Fatty acid labelling shows that lipid rafts are labelled preferentially with saturated fatty acids while the rest of the plasma membrane incorporates mostly long-chained polyunsaturated fatty acids. To see whether the raft composition as measured by metabolic labelling of phospholipids is involved in T-cell activation, we investigated the production of sn-1,2-diacylglycerol (DAG) in CD3-activated cells. DAG production occurs within rafts, confirming previous demonstration of protein kinase C translocation into membrane microdomains. Our data demonstrate that raft disorganization by methyl-beta-cyclodextrin impairs both CD3-induced DAG production and changes in cytosolic Ca(2+) concentration. These lines of evidence support the conclusion that the major events in T-cell activation occur within or due to lipid rafts. PMID:11964165

  6. Comparative proteomics reveals abnormal binding of ATGL and dysferlin on lipid droplets from pressure overload-induced dysfunctional rat hearts

    PubMed Central

    Li, Linghai; Zhang, Huina; Wang, Weiyi; Hong, Yun; Wang, Jifeng; Zhang, Shuyan; Xu, Shimeng; Shu, Qingbo; Li, Juanfen; Yang, Fuquan; Zheng, Min; Qian, Zongjie; Liu, Pingsheng

    2016-01-01

    Excessive retention of neutral lipids in cardiac lipid droplets (LDs) is a common observation in cardiomyopathy. Thus, the systematic investigation of the cardiac LD proteome will help to dissect the underlying mechanisms linking cardiac steatosis and myocardial dysfunction. Here, after isolation of LDs from normal and dysfunctional Sprague-Dawley rat hearts, we identified 752 heart-associated LD proteins using iTRAQ quantitative proteomic method, including 451 proteins previously unreported on LDs. The most noteworthy finding was the identification of the membrane resealing protein, dysferlin. An analysis of dysferlin truncation mutants indicated that its C2 domain was responsible for its LD localization. Quantitative proteomic results further determined that 27 proteins were increased and 16 proteins were decreased in LDs from post pressure overload-induced dysfunctional hearts, compared with normal hearts. Notably, adipose triacylglycerol lipase (ATGL) was dramatically decreased and dysferlin was substantially increased on dysfunctional cardiac LDs. This study for the first time reveals the dataset of the heart LD proteome in healthy tissue and the variation of it under cardiac dysfunction. These findings highlight an association between the altered LD protein localization of dysferlin and ATGL and myocardial dysfunction. PMID:26795240

  7. Comparative proteomics reveals abnormal binding of ATGL and dysferlin on lipid droplets from pressure overload-induced dysfunctional rat hearts.

    PubMed

    Li, Linghai; Zhang, Huina; Wang, Weiyi; Hong, Yun; Wang, Jifeng; Zhang, Shuyan; Xu, Shimeng; Shu, Qingbo; Li, Juanfen; Yang, Fuquan; Zheng, Min; Qian, Zongjie; Liu, Pingsheng

    2016-01-01

    Excessive retention of neutral lipids in cardiac lipid droplets (LDs) is a common observation in cardiomyopathy. Thus, the systematic investigation of the cardiac LD proteome will help to dissect the underlying mechanisms linking cardiac steatosis and myocardial dysfunction. Here, after isolation of LDs from normal and dysfunctional Sprague-Dawley rat hearts, we identified 752 heart-associated LD proteins using iTRAQ quantitative proteomic method, including 451 proteins previously unreported on LDs. The most noteworthy finding was the identification of the membrane resealing protein, dysferlin. An analysis of dysferlin truncation mutants indicated that its C2 domain was responsible for its LD localization. Quantitative proteomic results further determined that 27 proteins were increased and 16 proteins were decreased in LDs from post pressure overload-induced dysfunctional hearts, compared with normal hearts. Notably, adipose triacylglycerol lipase (ATGL) was dramatically decreased and dysferlin was substantially increased on dysfunctional cardiac LDs. This study for the first time reveals the dataset of the heart LD proteome in healthy tissue and the variation of it under cardiac dysfunction. These findings highlight an association between the altered LD protein localization of dysferlin and ATGL and myocardial dysfunction. PMID:26795240

  8. Atomistic MD simulation reveals the mechanism by which CETP penetrates into HDL enabling lipid transfer from HDL to CETP

    PubMed Central

    Cilpa-Karhu, Geraldine; Jauhiainen, Matti; Riekkola, Marja-Liisa

    2015-01-01

    Inhibition of cholesterol ester transfer protein (CETP), a protein mediating transfer of neutral lipids between lipoproteins, has been proposed as a means to elevate atheroprotective HDL subpopulations and thereby reduce atherosclerosis. However, off-target and adverse effects of the inhibition have raised doubts about the molecular mechanism of CETP-HDL interaction. Recent experimental findings have demonstrated the penetration of CETP into HDL. However, atomic level resolution of CETP penetration into HDL, a prerequisite for a better understanding of CETP functionality and HDL atheroprotection, is missing. We constructed an HDL particle that mimics the actual human HDL mass composition and investigated for the first time, by large-scale atomistic molecular dynamics, the interaction of an upright CETP with a human HDL-mimicking model. The results demonstrated how CETP can penetrate the HDL particle surface, with the formation of an opening in the N barrel domain end of CETP, put in evidence the major anchoring role of a tryptophan-rich region of this domain, and unveiled the presence of a phenylalanine barrier controlling further access of HDL-derived lipids to the tunnel of CETP. The findings reveal novel atomistic details of the CETP-HDL interaction mechanism and can provide new insight into therapeutic strategies. PMID:25424006

  9. A Systems Biology Approach Reveals the Role of a Novel Methyltransferase in Response to Chemical Stress and Lipid Homeostasis

    PubMed Central

    Lissina, Elena; Young, Brian; Urbanus, Malene L.; Guan, Xue Li; Lowenson, Jonathan; Hoon, Shawn; Baryshnikova, Anastasia; Riezman, Isabelle; Michaut, Magali; Riezman, Howard; Cowen, Leah E.; Wenk, Markus R.; Clarke, Steven G.; Giaever, Guri; Nislow, Corey

    2011-01-01

    Using small molecule probes to understand gene function is an attractive approach that allows functional characterization of genes that are dispensable in standard laboratory conditions and provides insight into the mode of action of these compounds. Using chemogenomic assays we previously identified yeast Crg1, an uncharacterized SAM-dependent methyltransferase, as a novel interactor of the protein phosphatase inhibitor cantharidin. In this study we used a combinatorial approach that exploits contemporary high-throughput techniques available in Saccharomyces cerevisiae combined with rigorous biological follow-up to characterize the interaction of Crg1 with cantharidin. Biochemical analysis of this enzyme followed by a systematic analysis of the interactome and lipidome of CRG1 mutants revealed that Crg1, a stress-responsive SAM-dependent methyltransferase, methylates cantharidin in vitro. Chemogenomic assays uncovered that lipid-related processes are essential for cantharidin resistance in cells sensitized by deletion of the CRG1 gene. Lipidome-wide analysis of mutants further showed that cantharidin induces alterations in glycerophospholipid and sphingolipid abundance in a Crg1-dependent manner. We propose that Crg1 is a small molecule methyltransferase important for maintaining lipid homeostasis in response to drug perturbation. This approach demonstrates the value of combining chemical genomics with other systems-based methods for characterizing proteins and elucidating previously unknown mechanisms of action of small molecule inhibitors. PMID:22028670

  10. Human Immunodeficiency Virus Type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains

    PubMed Central

    Brügger, Britta; Krautkrämer, Ellen; Tibroni, Nadine; Munte, Claudia E; Rauch, Susanne; Leibrecht, Iris; Glass, Bärbel; Breuer, Sebastian; Geyer, Matthias; Kräusslich, Hans-Georg; Kalbitzer, Hans Robert; Wieland, Felix T; Fackler, Oliver T

    2007-01-01

    Background The Nef protein of Human Immunodeficiency Viruses optimizes viral spread in the infected host by manipulating cellular transport and signal transduction machineries. Nef also boosts the infectivity of HIV particles by an unknown mechanism. Recent studies suggested a correlation between the association of Nef with lipid raft microdomains and its positive effects on virion infectivity. Furthermore, the lipidome analysis of HIV-1 particles revealed a marked enrichment of classical raft lipids and thus identified HIV-1 virions as an example for naturally occurring membrane microdomains. Since Nef modulates the protein composition and function of membrane microdomains we tested here if Nef also has the propensity to alter microdomain lipid composition. Results Quantitative mass spectrometric lipidome analysis of highly purified HIV-1 particles revealed that the presence of Nef during virus production from T lymphocytes enforced their raft character via a significant reduction of polyunsaturated phosphatidylcholine species and a specific enrichment of sphingomyelin. In contrast, Nef did not significantly affect virion levels of phosphoglycerolipids or cholesterol. The observed alterations in virion lipid composition were insufficient to mediate Nef's effect on particle infectivity and Nef augmented virion infectivity independently of whether virus entry was targeted to or excluded from membrane microdomains. However, altered lipid compositions similar to those observed in virions were also detected in detergent-resistant membrane preparations of virus producing cells. Conclusion Nef alters not only the proteome but also the lipid composition of host cell microdomains. This novel activity represents a previously unrecognized mechanism by which Nef could manipulate HIV-1 target cells to facilitate virus propagation in vivo. PMID:17908312

  11. Drop floating on a granular raft

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

    When a droplet comes in contact with a bath of the same liquid, it coalesces to minimize the surface energy. This phenomenon reduces emulsion stability and is usually fought with surfactant molecules. Another way to slow down coalescence is to use colloidal solid particles. In this case the particles spontaneously migrate to the interface to form ``Pickering'' emulsions and act as a barrier between droplets. Here we use dense, large particles (~ 500 μm) which form a monolayer at an oil/water interface that we call a granular raft. When a droplet is placed on top of such a raft, for a given set of particle properties (contact angle/size), the raft prevents coalescence indefinitely. However, in contrast to what happens when a droplet is placed on a hydrophobic surface and never wets the surface, here the droplet is strongly anchored to the raft and deforms it. We will use this specific configuration to probe the mechanical response of the granular raft: by controlling the droplet volume we can impose tensile or compressive stresses. Finally we will show that the drop, spherical at first, slowly takes a more complex shape as it's volume increases. This shape is not reversible as the drop volume is decreased. The drop can become oblate or prolate with wrinkling of the raft.

  12. Remorin, a solanaceae protein resident in membrane rafts and plasmodesmata, impairs potato virus X movement.

    PubMed

    Raffaele, Sylvain; Bayer, Emmanuelle; Lafarge, David; Cluzet, Stéphanie; German Retana, Sylvie; Boubekeur, Tamy; Leborgne-Castel, Nathalie; Carde, Jean-Pierre; Lherminier, Jeannine; Noirot, Elodie; Satiat-Jeunemaître, Béatrice; Laroche-Traineau, Jeanny; Moreau, Patrick; Ott, Thomas; Maule, Andrew J; Reymond, Philippe; Simon-Plas, Françoise; Farmer, Edward E; Bessoule, Jean-Jacques; Mongrand, Sébastien

    2009-05-01

    Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in approximately 70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane. PMID:19470590

  13. Revealing Transient Interactions between Phosphatidylinositol-specific Phospholipase C and Phosphatidylcholine--Rich Lipid Vesicles

    NASA Astrophysics Data System (ADS)

    Yang, Boqian; He, Tao; Grauffel, Cédric; Reuter, Nathalie; Roberts, Mary; Gershenson, Anne

    2013-03-01

    Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes transiently interact with target membranes. Previous fluorescence correlation spectroscopy (FCS) experiments showed that Bacillus thuringiensis PI-PLC specifically binds to phosphatidylcholine (PC)-rich membranes and preferentially interacts with unilamellar vesicles that show larger curvature. Mutagenesis studies combined with FCS measurements of binding affinity highlighted the importance of interfacial PI-PLC tyrosines in the PC specificity. All-atom molecular dynamics simulations of PI-PLC performed in the presence of a PC membrane indicate these tyrosines are involved in specific cation-pi interactions with choline headgroups. To further understand those transient interactions between PI-PLC and PC-rich vesicles, we monitor single fluorescently labeled PI-PLC proteins as they cycle on and off surface-tethered small unilamellar vesicles using total internal reflection fluorescent microscopy. The residence times on vesicles along with vesicle size information, based on vesicle fluorescence intensity, reveal the time scales of PI-PLC membrane interactions as well as the curvature dependence. The PC specificity and the vesicle curvature dependence of this PI-PLC/membrane interaction provide insight into how the interface modulates protein-membrane interactions. This work was supported by the National Institute of General Medical Science of the National Institutes of Health (R01GM060418).

  14. Metabolomic signatures in lipid-loaded HepaRGs reveal pathways involved in steatotic progression

    PubMed Central

    Brown, MV; Compton, SA; Milburn, MV; Lawton, KA; Cheatham, B

    2013-01-01

    Objectives Non-alcoholic fatty liver disease (NAFLD) describes a spectrum of disorders including simple steatosis, non-alcoholic steatohepatitis, fibrosis, and cirrhosis. With the increased prevalence of obesity, and consequently NAFLD, there is a need for novel therapeutics in this area. To facilitate this effort, we developed a cellular model of hepatic steatosis using HepaRG cells and determined the resulting biochemical alterations. Design and methods Using global metabolomic profiling, by means of a novel metabolite extraction procedure, we examined the metabolic profiles in response to the saturated fatty acid palmitate, and a mixture of saturated and unsaturated fatty acids, palmitate and oleate (1:2). Results We observed elevated levels of the branched chain amino acids, TCA cycle intermediates, sphingosine and acylcarnitines, and reduced levels of carnitine in the steatotic HepaRG model with both palmitate and palmitate:oleate treatments. In addition, palmitate-induced steatotic cells selectively displayed elevated levels of diacylglycerols and monoacylglycerols, as well as altered bile acid metabolism. Conclusion This global metabolomics approach reveals biochemical changes in pathways important in the transition to hepatic steatosis including insulin resistance, altered mitochondrial metabolism, and oxidative stress. Moreover, our data demonstrate the utility of this in vitro model for investigating mechanisms of steatotic progression, insulin resistance and lipotoxicity in NAFLD. PMID:23512965

  15. Norepinephrine and endothelin activate diacylglycerol kinases in caveolae/rafts of rat mesenteric arteries: agonist-specific role of PI3-kinase.

    PubMed

    Clarke, Christopher J; Ohanian, Vasken; Ohanian, Jacqueline

    2007-05-01

    The phosphatidylinositol (PI) signaling pathway mediates norepinephrine (NE)- and endothelin-1 (ET-1)-stimulated vascular smooth muscle contraction through an inositol-trisphosphate-induced rise in intracellular calcium and diacylglycerol (DG) activation of protein kinase C (PKC). Subsequent activation of DG kinases (DGKs) metabolizes DG to phosphatidic acid (PA), potentially regulating PKC activity. Because precise regulation and spatial restriction of the PI pathway is necessary for specificity, we have investigated whether this occurs within caveolae/rafts, specialized plasma membrane microdomains implicated in vascular smooth muscle contraction. We show that components of the PI signaling cascade-phosphatidylinositol 4,5-bisphosphate (PIP(2)), PA, and DGK-theta are present in caveolae/rafts prepared from rat mesenteric small arteries. Stimulation with NE or ET-1 induced [(33)P]PIP(2) hydrolysis solely within caveolae/rafts. NE stimulated an increase in DGK activity in caveolae/rafts alone, whereas ET-1 activated DGK in caveolae/rafts and noncaveolae/rafts; however, [(33)P]PA increased in all fractions with both agonists. Previously, we reported that NE activated DGK-theta in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner; here, we describe PI3-kinase-dependent DGK activation and [(33)P]PA production in caveolae/rafts in response to NE but not ET-1. Additionally, PKB, a potential activator of DGK-theta, translocated to caveolae/rafts in response to NE but not ET-1, and PI3-kinase inhibition prevented this. Furthermore, PI3-kinase inhibition reduced the sensitivity of contraction to NE but not ET-1. Our study shows that caveolae/rafts are major sites of vasoconstrictor hormone activation of the PI pathway in intact small arteries and suggest a link between lipid signaling events within caveolae/rafts and contraction. PMID:17208990

  16. Analysis of lipids reveals differences between 'Mycobacterium habana' and Mycobacterium simiae.

    PubMed

    Mederos, L M; Valdivia, J A; Sempere, M A; Valero-Guillén, P L

    1998-05-01

    Fatty and mycolic acids and the pattern of glycolipids were studied in a collection of 34 strains of 'Mycobacterium habana' and in two strains of Mycobacterium simiae. Major glycolipids of these micro-organisms were assigned to the glycopeptidolipid (GPL) structural type, but both mycobacteria differed in the patterns obtained by TLC. The strains of 'M. habana' were separated into four groups (A-D), taking into account the presence or absence of several polar GPLs: group A contained GPL-I, GPL-II and GPL-III; group B contained GPL-I, GPL-II', GPL-II and GPL-III; group C contained GPL-II', GPL-II and GPL-III; group D did not contain any of these compounds. Fatty acids of both bacteria were similar, and ranged from 14 to 26 carbon atoms, hexadecanoic, octadecenoic and tuberculostearic acids being predominant. Mycolic acids were also similar by TLC and HPLC, and consisted of alpha-, alpha'- and ketomycolates. Partial structural analysis by MS carried out in strains 'M. habana' TMC 5135 and M. simiae ATCC 25275T revealed that alpha- and ketomycolates ranged, in general, from 79 to 87 carbon atoms, and alpha'-mycolates from 58 to 67 carbon atoms. The alpha- and ketomycolates belonged to several structural series, and minor variations were found between the two strain examined. The data obtained justified the synonymy between 'M. habana' and M. simiae but indicated, in turn, that the former can be distinguished on the basis of GPL analysis. Most strains of 'M. habana' can be defined by the presence of GPL-II and GPL-III, a finding that could be useful in the quality control of potential vaccine strains. PMID:9611792

  17. ATR–FTIR spectroscopy reveals involvement of lipids and proteins of intact pea pollen grains to heat stress tolerance

    PubMed Central

    Lahlali, Rachid; Jiang, Yunfei; Kumar, Saroj; Karunakaran, Chithra; Liu, Xia; Borondics, Ferenc; Hallin, Emil; Bueckert, Rosalind

    2014-01-01

    With climate change, pea will be more frequently subjected to heat stress in semi-arid regions like Saskatchewan during flowering. The pollen germination percentage of two pea cultivars was reduced by heat stress (36°C) with an important decrease in cultivar ‘CDC Golden’ compared to ‘CDC Sage.’ Lipids, protein and other pollen coat compositions of whole intact pollen grains of both pea cultivars were investigated using mid infrared (mid-IR) attenuated total reflectance (ATR)–Fourier transform infrared (FTIR) spectroscopy. Curve fitting of ATR absorbance spectra in the protein region enabled estimation and comparison of different protein secondary structures between the two cultivars. CDC Sage had relatively greater amounts of α-helical structures (48.6–43.6%; band at 1654 cm-1) and smaller amounts of β-sheets (41.3–46%) than CDC Golden. The CDC Golden had higher amounts of β-sheets (46.3–51.7%) compared to α-helical structures (35.3–36.2%). Further, heat stress resulted in prominent changes in the symmetrical and asymmetrical CH2 bands from lipid acyl chain, ester carbonyl band, and carbohydrate region. The intensity of asymmetric and symmetric CH2 vibration of heat stressed CDC Golden was reduced considerably in comparison to the control and the decrease was higher compared to CDC Sage. In addition, CDC Golden showed an increase in intensity at the oxidative band of 3015 cm-1. These results reveal that the whole pollen grains of both pea cultivars responded differently to heat stress. The tolerance of CDC Sage to heat stress (expressed as pollen germination percentage) may be due to its protein richness with α-helical structures which would protect against the destructive effects of dehydration due to heat stress. The low pollen germination percentage of CDC Golden after heat stress may be also due to its sensitivity to lipid changes due to heat stress. PMID:25566312

  18. Ozone-Induced Dissociation of Conjugated Lipids Reveals Significant Reaction Rate Enhancements and Characteristic Odd-Electron Product Ions

    NASA Astrophysics Data System (ADS)

    Pham, Huong T.; Maccarone, Alan T.; Campbell, J. Larry; Mitchell, Todd W.; Blanksby, Stephen J.

    2013-02-01

    Ozone-induced dissociation (OzID) is an alternative ion activation method that relies on the gas phase ion-molecule reaction between a mass-selected target ion and ozone in an ion trap mass spectrometer. Herein, we evaluated the performance of OzID for both the structural elucidation and selective detection of conjugated carbon-carbon double bond motifs within lipids. The relative reactivity trends for [M + X]+ ions (where X = Li, Na, K) formed via electrospray ionization (ESI) of conjugated versus nonconjugated fatty acid methyl esters (FAMEs) were examined using two different OzID-enabled linear ion-trap mass spectrometers. Compared with nonconjugated analogues, FAMEs derived from conjugated linoleic acids were found to react up to 200 times faster and to yield characteristic radical cations. The significantly enhanced reactivity of conjugated isomers means that OzID product ions can be observed without invoking a reaction delay in the experimental sequence (i.e., trapping of ions in the presence of ozone is not required). This possibility has been exploited to undertake neutral-loss scans on a triple quadrupole mass spectrometer targeting characteristic OzID transitions. Such analyses reveal the presence of conjugated double bonds in lipids extracted from selected foodstuffs. Finally, by benchmarking of the absolute ozone concentration inside the ion trap, second order rate constants for the gas phase reactions between unsaturated organic ions and ozone were obtained. These results demonstrate a significant influence of the adducting metal on reaction rate constants in the fashion Li > Na > K.

  19. Amyloidβ Peptides in interaction with raft-mime model membranes: a neutron reflectivity insight

    PubMed Central

    Rondelli, Valeria; Brocca, Paola; Motta, Simona; Messa, Massimo; Colombo, Laura; Salmona, Mario; Fragneto, Giovanna; Cantù, Laura; Del Favero, Elena

    2016-01-01

    The role of first-stage β–amyloid aggregation in the development of the Alzheimer disease, is widely accepted but still unclear. Intimate interaction with the cell membrane is invoked. We designed Neutron Reflectometry experiments to reveal the existence and extent of the interaction between β–amyloid (Aβ) peptides and a lone customized biomimetic membrane, and their dependence on the aggregation state of the peptide. The membrane, asymmetrically containing phospholipids, GM1 and cholesterol in biosimilar proportion, is a model for a raft, a putative site for amyloid-cell membrane interaction. We found that the structured-oligomer of Aβ(1-42), its most acknowledged membrane-active state, is embedded as such into the external leaflet of the membrane. Conversely, the Aβ(1-42) unstructured early-oligomers deeply penetrate the membrane, likely mimicking the interaction at neuronal cell surfaces, when the Aβ(1-42) is cleaved from APP protein and the membrane constitutes a template for its further structural evolution. Moreover, the smaller Aβ(1-6) fragment, the N-terminal portion of Aβ, was also used. Aβ N-terminal is usually considered as involved in oligomer stabilization but not in the peptide-membrane interaction. Instead, it was seen to remove lipids from the bilayer, thus suggesting its role, once in the whole peptide, in membrane leakage, favouring peptide recruitment. PMID:26880066

  20. Amyloid-β peptides in interaction with raft-mime model membranes: a neutron reflectivity insight.

    PubMed

    Rondelli, Valeria; Brocca, Paola; Motta, Simona; Messa, Massimo; Colombo, Laura; Salmona, Mario; Fragneto, Giovanna; Cantù, Laura; Del Favero, Elena

    2016-01-01

    The role of first-stage β-amyloid aggregation in the development of the Alzheimer disease, is widely accepted but still unclear. Intimate interaction with the cell membrane is invoked. We designed Neutron Reflectometry experiments to reveal the existence and extent of the interaction between β-amyloid (Aβ) peptides and a lone customized biomimetic membrane, and their dependence on the aggregation state of the peptide. The membrane, asymmetrically containing phospholipids, GM1 and cholesterol in biosimilar proportion, is a model for a raft, a putative site for amyloid-cell membrane interaction. We found that the structured-oligomer of Aβ(1-42), its most acknowledged membrane-active state, is embedded as such into the external leaflet of the membrane. Conversely, the Aβ(1-42) unstructured early-oligomers deeply penetrate the membrane, likely mimicking the interaction at neuronal cell surfaces, when the Aβ(1-42) is cleaved from APP protein and the membrane constitutes a template for its further structural evolution. Moreover, the smaller Aβ(1-6) fragment, the N-terminal portion of Aβ, was also used. Aβ N-terminal is usually considered as involved in oligomer stabilization but not in the peptide-membrane interaction. Instead, it was seen to remove lipids from the bilayer, thus suggesting its role, once in the whole peptide, in membrane leakage, favouring peptide recruitment. PMID:26880066

  1. An Optimized Histochemical Method to Assess Skeletal Muscle Glycogen and Lipid Stores Reveals Two Metabolically Distinct Populations of Type I Muscle Fibers

    PubMed Central

    Prats, Clara; Gomez-Cabello, Alba; Nordby, Pernille; Andersen, Jesper L.; Helge, Jørn W.; Dela, Flemming; Baba, Otto; Ploug, Thorkil

    2013-01-01

    Skeletal muscle energy metabolism has been a research focus of physiologists for more than a century. Yet, how the use of intramuscular carbohydrate and lipid energy stores are coordinated during different types of exercise remains a subject of debate. Controversy arises from contradicting data from numerous studies, which used different methodological approaches. Here we review the “pros and cons” of previously used histochemical methods and describe an optimized method to ensure the preservation and specificity of detection of both intramyocellular carbohydrate and lipid stores. For optimal preservation of muscle energy stores, air drying cryosections or cycles of freezing-thawing need to be avoided. Furthermore, optimization of the imaging settings in order to specifically image intracellular lipid droplets stained with oil red O or Bodipy-493/503 is shown. When co-staining lipid droplets with associated proteins, Bodipy-493/503 should be the dye of choice, since oil red O creates precipitates on the lipid droplets blocking the light. In order to increase the specificity of glycogen stain, an antibody against glycogen is used. The resulting method reveals the existence of two metabolically distinct myosin heavy chain I expressing fibers: I-1 fibers have a smaller crossectional area, a higher density of lipid droplets, and a tendency to lower glycogen content compared to I-2 fibers. Type I-2 fibers have similar lipid content than IIA. Exhaustive exercise lead to glycogen depletion in type IIA and IIX fibers, a reduction in lipid droplets density in both type I-1 and I-2 fibers, and a decrease in the size of lipid droplets exclusively in type I-1 fibers. PMID:24204959

  2. Raft-based interactions of gangliosides with a GPI-anchored receptor.

    PubMed

    Komura, Naoko; Suzuki, Kenichi G N; Ando, Hiromune; Konishi, Miku; Koikeda, Machi; Imamura, Akihiro; Chadda, Rahul; Fujiwara, Takahiro K; Tsuboi, Hisae; Sheng, Ren; Cho, Wonhwa; Furukawa, Koichi; Furukawa, Keiko; Yamauchi, Yoshio; Ishida, Hideharu; Kusumi, Akihiro; Kiso, Makoto

    2016-06-01

    Gangliosides, glycosphingolipids containing one or more sialic acid(s) in the glyco-chain, are involved in various important physiological and pathological processes in the plasma membrane. However, their exact functions are poorly understood, primarily because of the scarcity of suitable fluorescent ganglioside analogs. Here, we developed methods for systematically synthesizing analogs that behave like their native counterparts in regard to partitioning into raft-related membrane domains or preparations. Single-fluorescent-molecule imaging in the live-cell plasma membrane revealed the clear but transient colocalization and codiffusion of fluorescent ganglioside analogs with a fluorescently labeled glycosylphosphatidylinisotol (GPI)-anchored protein, human CD59, with lifetimes of 12 ms for CD59 monomers, 40 ms for CD59's transient homodimer rafts in quiescent cells, and 48 ms for engaged-CD59-cluster rafts, in cholesterol- and GPI-anchoring-dependent manners. The ganglioside molecules were always mobile in quiescent cells. These results show that gangliosides continually and dynamically exchange between raft domains and the bulk domain, indicating that raft domains are dynamic entities. PMID:27043189

  3. 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

    SciTech Connect

    De Gregorio, Francesca; Pellegrino, Mario; Picchietti, Simona; Belardinelli, Maria C.; Taddei, Anna Rita; Fausto, Anna Maria; Rossi, Mario; Maggio, Roberto; Giorgi, Franco

    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 the 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 receptor

  4. Single cell synchrotron FT-IR microspectroscopy reveals a link between neutral lipid and storage carbohydrate fluxes in S. cerevisiae.

    PubMed

    Jamme, Frédéric; Vindigni, Jean-David; Méchin, Valérie; Cherifi, Tamazight; Chardot, Thierry; Froissard, Marine

    2013-01-01

    In most organisms, storage lipids are packaged into specialized structures called lipid droplets. These contain a core of neutral lipids surrounded by a monolayer of phospholipids, and various proteins which vary depending on the species. Hydrophobic structural proteins stabilize the interface between the lipid core and aqueous cellular environment (perilipin family of proteins, apolipoproteins, oleosins). We developed a genetic approach using heterologous expression in Saccharomyces cerevisiae of the Arabidopsis thaliana lipid droplet oleosin and caleosin proteins AtOle1 and AtClo1. These transformed yeasts overaccumulate lipid droplets, leading to a specific increase in storage lipids. The phenotype of these cells was explored using synchrotron FT-IR microspectroscopy to investigate the dynamics of lipid storage and cellular carbon fluxes reflected as changes in spectral fingerprints. Multivariate statistical analysis of the data showed a clear effect on storage carbohydrates and more specifically, a decrease in glycogen in our modified strains. These observations were confirmed by biochemical quantification of the storage carbohydrates glycogen and trehalose. Our results demonstrate that neutral lipid and storage carbohydrate fluxes are tightly connected and co-regulated. PMID:24040242

  5. Single Cell Synchrotron FT-IR Microspectroscopy Reveals a Link between Neutral Lipid and Storage Carbohydrate Fluxes in S. cerevisiae

    PubMed Central

    Jamme, Frédéric; Vindigni, Jean-David; Méchin, Valérie; Cherifi, Tamazight; Chardot, Thierry; Froissard, Marine

    2013-01-01

    In most organisms, storage lipids are packaged into specialized structures called lipid droplets. These contain a core of neutral lipids surrounded by a monolayer of phospholipids, and various proteins which vary depending on the species. Hydrophobic structural proteins stabilize the interface between the lipid core and aqueous cellular environment (perilipin family of proteins, apolipoproteins, oleosins). We developed a genetic approach using heterologous expression in Saccharomyces cerevisiae of the Arabidopsis thaliana lipid droplet oleosin and caleosin proteins AtOle1 and AtClo1. These transformed yeasts overaccumulate lipid droplets, leading to a specific increase in storage lipids. The phenotype of these cells was explored using synchrotron FT-IR microspectroscopy to investigate the dynamics of lipid storage and cellular carbon fluxes reflected as changes in spectral fingerprints. Multivariate statistical analysis of the data showed a clear effect on storage carbohydrates and more specifically, a decrease in glycogen in our modified strains. These observations were confirmed by biochemical quantification of the storage carbohydrates glycogen and trehalose. Our results demonstrate that neutral lipid and storage carbohydrate fluxes are tightly connected and co-regulated. PMID:24040242

  6. Systems Chemo-Biology and Transcriptomic Meta-Analysis Reveal the Molecular Roles of Bioactive Lipids in Cardiomyocyte Differentiation.

    PubMed

    de Faria Poloni, Joice; Bonatto, Diego

    2015-09-01

    Lipids, which are essential constituents of biological membranes, play structural and functional roles in the cell. In recent years, certain lipids have been identified as regulatory signaling molecules and have been termed "bioactive lipids". Subsequently, the importance of bioactive lipids in stem cell differentiation and cardiogenesis has gained increasing recognition. Therefore, the aim of this study was to identify the biological processes underlying murine cardiac differentiation and the mechanisms by which bioactive lipids affect these processes. For this purpose, a transcriptomic meta-analysis of microarray and RNA-seq data from murine stem cells undergoing cardiogenic differentiation was performed. The differentially expressed genes identified via this meta-analysis, as well as bioactive lipids, were evaluated using systems chemo-biology tools. These data indicated that bioactive lipids are associated with the regulation of cell motility, cell adhesion, cytoskeletal rearrangement, and gene expression. Moreover, bioactive lipids integrate the signaling pathways involved in cell migration, the secretion and remodeling of extracellular matrix components, and the establishment of the cardiac phenotype. In conclusion, this study provides new insights into the contribution of bioactive lipids to the induction of cellular responses to various stimuli, which may originate from the extracellular environment and morphogens, and the manner in which this contribution directly affects murine heart morphogenesis. PMID:25752681

  7. Raptor ecology of Raft River Valley, Idaho

    SciTech Connect

    Thurow, T.L.; White, C.M.; Howard, R.P.; Sullivan, J.F.

    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.

  8. Hedgehog signaling is a potent regulator of liver lipid metabolism and reveals a GLI-code associated with steatosis

    PubMed Central

    Matz-Soja, Madlen; Rennert, Christiane; Schönefeld, Kristin; Aleithe, Susanne; Boettger, Jan; Schmidt-Heck, Wolfgang; Weiss, Thomas S; Hovhannisyan, Amalya; Zellmer, Sebastian; Klöting, Nora; Schulz, Angela; Kratzsch, Jürgen; Guthke, Reinhardt; Gebhardt, Rolf

    2016-01-01

    Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries and is increasing in prevalence. The pathomechanisms, however, are poorly understood. This study assessed the unexpected role of the Hedgehog pathway in adult liver lipid metabolism. Using transgenic mice with conditional hepatocyte-specific deletion of Smoothened in adult mice, we showed that hepatocellular inhibition of Hedgehog signaling leads to steatosis by altering the abundance of the transcription factors GLI1 and GLI3. This steatotic 'Gli-code' caused the modulation of a complex network of lipogenic transcription factors and enzymes, including SREBP1 and PNPLA3, as demonstrated by microarray analysis and siRNA experiments and could be confirmed in other steatotic mouse models as well as in steatotic human livers. Conversely, activation of the Hedgehog pathway reversed the "Gli-code" and mitigated hepatic steatosis. Collectively, our results reveal that dysfunctions in the Hedgehog pathway play an important role in hepatic steatosis and beyond. DOI: http://dx.doi.org/10.7554/eLife.13308.001 PMID:27185526

  9. Hedgehog signaling is a potent regulator of liver lipid metabolism and reveals a GLI-code associated with steatosis.

    PubMed

    Matz-Soja, Madlen; Rennert, Christiane; Schönefeld, Kristin; Aleithe, Susanne; Boettger, Jan; Schmidt-Heck, Wolfgang; Weiss, Thomas S; Hovhannisyan, Amalya; Zellmer, Sebastian; Klöting, Nora; Schulz, Angela; Kratzsch, Jürgen; Guthke, Reinhardt; Gebhardt, Rolf

    2016-01-01

    Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries and is increasing in prevalence. The pathomechanisms, however, are poorly understood. This study assessed the unexpected role of the Hedgehog pathway in adult liver lipid metabolism. Using transgenic mice with conditional hepatocyte-specific deletion of Smoothened in adult mice, we showed that hepatocellular inhibition of Hedgehog signaling leads to steatosis by altering the abundance of the transcription factors GLI1 and GLI3. This steatotic 'Gli-code' caused the modulation of a complex network of lipogenic transcription factors and enzymes, including SREBP1 and PNPLA3, as demonstrated by microarray analysis and siRNA experiments and could be confirmed in other steatotic mouse models as well as in steatotic human livers. Conversely, activation of the Hedgehog pathway reversed the "Gli-code" and mitigated hepatic steatosis. Collectively, our results reveal that dysfunctions in the Hedgehog pathway play an important role in hepatic steatosis and beyond. PMID:27185526

  10. The nisin-lipid II complex reveals a pyrophosphate cage that provides a blueprint for novel antibiotics.

    PubMed

    Hsu, Shang-Te D; Breukink, Eefjan; Tischenko, Eugene; Lutters, Mandy A G; de Kruijff, Ben; Kaptein, Robert; Bonvin, Alexandre M J J; van Nuland, Nico A J

    2004-10-01

    The emerging antibiotics-resistance problem has underlined the urgent need for novel antimicrobial agents. Lantibiotics (lanthionine-containing antibiotics) are promising candidates to alleviate this problem. Nisin, a member of this family, has a unique pore-forming activity against bacteria. It binds to lipid II, the essential precursor of cell wall synthesis. As a result, the membrane permeabilization activity of nisin is increased by three orders of magnitude. Here we report the solution structure of the complex of nisin and lipid II. The structure shows a novel lipid II-binding motif in which the pyrophosphate moiety of lipid II is primarily coordinated by the N-terminal backbone amides of nisin via intermolecular hydrogen bonds. This cage structure provides a rationale for the conservation of the lanthionine rings among several lipid II-binding lantibiotics. The structure of the pyrophosphate cage offers a template for structure-based design of novel antibiotics. PMID:15361862

  11. De novo Assembly and Characterization of the Fruit Transcriptome of Idesia polycarpa Reveals Candidate Genes for Lipid Biosynthesis

    PubMed Central

    Li, Rong-Jun; Gao, Xiang; Li, Lin-Mao; Liu, Xiu-Lin; Wang, Zhou-Ya; Lü, Shi-you

    2016-01-01

    Idesia polycarpa, is a valuable oilseed-producing tree of the Flacourtiaceae family that has the potential to fulfill edible oil production and is also a possible biofuel feedstock. The fruit is unique in that it contains both saturated and unsaturated lipids present in pericarp and seed, respectively. However, triglyceride synthesis and storage in tissues outside of the seeds has been poorly studied in previous researches. To gain insight into the unique properties of I. polycarpa fruit lipid synthesis, biochemical, and transcriptomic approaches were used to compare the lipid accumulation between pericarp and seed of the fruit. Lipid accumulation rates, final lipid content and composition were significantly different between two tissues. Furthermore, we described the annotated transcriptome assembly and differential gene expression analysis generated from the pericarp and seed tissues. The data allowed the identification of distinct candidate genes and reconstruction of lipid pathways, which may explain the differences of oil synthesis between the two tissues. The results may be useful for engineering alternative pathways for lipid production in non-seed or vegetative tissues. PMID:27375655

  12. De novo Assembly and Characterization of the Fruit Transcriptome of Idesia polycarpa Reveals Candidate Genes for Lipid Biosynthesis.

    PubMed

    Li, Rong-Jun; Gao, Xiang; Li, Lin-Mao; Liu, Xiu-Lin; Wang, Zhou-Ya; Lü, Shi-You

    2016-01-01

    Idesia polycarpa, is a valuable oilseed-producing tree of the Flacourtiaceae family that has the potential to fulfill edible oil production and is also a possible biofuel feedstock. The fruit is unique in that it contains both saturated and unsaturated lipids present in pericarp and seed, respectively. However, triglyceride synthesis and storage in tissues outside of the seeds has been poorly studied in previous researches. To gain insight into the unique properties of I. polycarpa fruit lipid synthesis, biochemical, and transcriptomic approaches were used to compare the lipid accumulation between pericarp and seed of the fruit. Lipid accumulation rates, final lipid content and composition were significantly different between two tissues. Furthermore, we described the annotated transcriptome assembly and differential gene expression analysis generated from the pericarp and seed tissues. The data allowed the identification of distinct candidate genes and reconstruction of lipid pathways, which may explain the differences of oil synthesis between the two tissues. The results may be useful for engineering alternative pathways for lipid production in non-seed or vegetative tissues. PMID:27375655

  13. Order of lipid phases in model and plasma membranes

    PubMed Central

    Kaiser, Hermann-Josef; Lingwood, Daniel; Levental, Ilya; Sampaio, Julio L.; Kalvodova, Lucie; Rajendran, Lawrence; Simons, Kai

    2009-01-01

    Lipid rafts are nanoscopic assemblies of sphingolipids, cholesterol, and specific membrane proteins that contribute to lateral heterogeneity in eukaryotic membranes. Separation of artificial membranes into liquid-ordered (Lo) and liquid-disordered phases is regarded as a common model for this compartmentalization. However, tight lipid packing in Lo phases seems to conflict with efficient partitioning of raft-associated transmembrane (TM) proteins. To assess membrane order as a component of raft organization, we performed fluorescence spectroscopy and microscopy with the membrane probes Laurdan and C-laurdan. First, we assessed lipid packing in model membranes of various compositions and found cholesterol and acyl chain dependence of membrane order. Then we probed cell membranes by using two novel systems that exhibit inducible phase separation: giant plasma membrane vesicles [Baumgart et al. (2007) Proc Natl Acad Sci USA 104:3165–3170] and plasma membrane spheres. Notably, only the latter support selective inclusion of raft TM proteins with the ganglioside GM1 into one phase. We measured comparable small differences in order between the separated phases of both biomembranes. Lateral packing in the ordered phase of giant plasma membrane vesicles resembled the Lo domain of model membranes, whereas the GM1 phase in plasma membrane spheres exhibited considerably lower order, consistent with different partitioning of lipid and TM protein markers. Thus, lipid-mediated coalescence of the GM1 raft domain seems to be distinct from the formation of a Lo phase, suggesting additional interactions between proteins and lipids to be effective. PMID:19805351

  14. Dynamics of submersible mussel rafts in waves and current

    NASA Astrophysics Data System (ADS)

    Wang, Xin-xin; Swift, M. Robinson; Dewhurst, Tobias; Tsukrov, Igor; Celikkol, Barbaros; Newell, Carter

    2015-06-01

    To investigate the dynamics of submersible mussel rafts, the finite element program Aqua-FE™, developed by the University of New Hampshire (UNH), was applied to rafts moored at the surface and submerged. The submerged configuration is used to reduce wave forcing and to avoid contact with floating ice during winters in northern waters. Each raft consists of three pontoons connected by a grid framework. Rafts are intended to support densely spaced mussel ropes hung from the framework. When submerged, the pontoons are flooded, and the raft is held vertically by floats attached by lines. The computer models were developed in Aqua-FE™ to simulate the effects of waves and current. They were validated by comparison with wave tank results by use of a 1/10 scale raft physical model. Comparisons showed good agreement for the important heave (vertical) and pitch (rotational) motions, though there was a tendency towards conservative results for wave and current drag. Full-scale simulations of surface and submerged single raft and two rafts connected in tandem were performed. Submerged raft wave response was found to be reduced relative to that at the surface for both the single and two-raft configurations. In particular, the vertical motion of mussel rope connection points was significantly reduced by submergence, resulting in reduced potential for mussel drop-off. For example, the maximum vertical velocities of mussel rope attachment points in the submerged two raft case were 7%-20% of the corresponding velocities when at the surface.

  15. Chemical Genetics Approach Reveals Importance of cAMP and MAP Kinase Signaling to Lipid and Carotenoid Biosynthesis in Microalgae.

    PubMed

    Choi, Yoon-E; Rhee, Jin-Kyu; Kim, Hyun-Soo; Ahn, Joon-Woo; Hwang, Hyemin; Yang, Ji-Won

    2015-05-01

    In this study, we attempted to understand signaling pathways behind lipid biosynthesis by employing a chemical genetics approach based on small molecule inhibitors. Specific signaling inhibitors of MAP kinase or modulators of cAMP signaling were selected to evaluate the functional roles of each of the key signaling pathways in three different microalgal species: Chlamydomonas reinhardtii, Chlorella vulgaris, and Haematococcus pluvialis. Our results clearly indicate that cAMP signaling pathways are indeed positively associated with microalgal lipid biosynthesis. In contrast, MAP kinase pathways in three microalgal species are all negatively implicated in both lipid and carotenoid biosynthesis. PMID:25563422

  16. Dissecting lipid metabolism in meibomian glands of humans and mice: An integrative study reveals a network of metabolic reactions not duplicated in other tissues.

    PubMed

    Butovich, Igor A; McMahon, Anne; Wojtowicz, Jadwiga C; Lin, Feng; Mancini, Ronald; Itani, Kamel

    2016-06-01

    Lipids comprise the bulk of the meibomian gland secretion (meibum) which is produced by meibocytes. Complex arrays of lipogenic reactions in meibomian glands, which we collectively call meibogenesis, have not been explored on a molecular level yet. Our goals were to elucidate the possible biosynthetic pathways that underlie the generation of meibum, reveal similarities in, and differences between, lipid metabolism in meibomian glands and other organs and tissues, and integrate meibomian gland studies into the field of general metabolomics. Specifically, we have conducted detailed analyses of human and mouse specimens using genomic, immunohistochemical, and lipidomic approaches. Among equally highly expressed genes found in meibomian glands of both species were those related to fatty acid elongation, branching, desaturation, esterification, reduction of fatty acids to alcohols, and cholesterol biosynthesis. Importantly, corresponding lipid products were detected in meibum of both species using lipidomic approaches. For the first time, a cohesive, unifying biosynthetic scheme that connects genomic, lipidomic, and immunohistochemical observations is outlined and discussed. PMID:27032494

  17. MALDI-TOF mass spectrometry imaging reveals molecular level changes in ultrahigh molecular weight polyethylene joint implants in correlation with lipid adsorption.

    PubMed

    Fröhlich, Sophie M; Archodoulaki, Vasiliki-Maria; Allmaier, Günter; Marchetti-Deschmann, Martina

    2014-10-01

    Ultrahigh molecular weight polyethylene (PE-UHMW), a material with high biocompatibility and excellent mechanical properties, is among the most commonly used materials for acetabular cup replacement in artificial joint systems. It is assumed that the interaction with synovial fluid in the biocompartment leads to significant changes relevant to material failure. In addition to hyaluronic acid, lipids are particularly relevant for lubrication in an articulating process. This study investigates synovial lipid adsorption on two different PE-UHMW materials (GUR-1050 and vitamin E-doped) in an in vitro model system by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry imaging (MSI). Lipids were identified by high performance thin layer chromatography (HP-TLC) and tandem mass spectrometry (MS/MS) analysis, with an analytical focus on phospholipids and cholesterol, both being species of high importance for lubrication. Scanning electron microscopy (SEM) analysis was applied in the study to correlate molecular information with PE-UHMW material qualities. It is demonstrated that lipid adsorption preferentially occurs in rough or oxidized polymer regions. Polymer modifications were colocalized with adsorbed lipids and found with high density in regions identified by SEM. Explanted, the in vivo polymer material showed comparable and even more obvious polymer damage and lipid adsorption when compared with the static in vitro model. A three-dimensional reconstruction of MSI data from consecutive PE-UHMW slices reveals detailed information about the diffusion process of lipids in the acetabular cup and provides, for the first time, a promising starting point for future studies correlating molecular information with commonly used techniques for material analysis (e.g., Fourier-transform infrared spectroscopy, nanoindentation). PMID:25215499

  18. Composition of Hydrothermal Vent Microbial Communities as Revealed by Analyses of Signature Lipids, Stable Carbon Isotopes and Aquificales Cultures

    NASA Technical Reports Server (NTRS)

    Jahnke, L. L.; Eder, W.; Huber, Robert; Hinrichs, K-U.; Hayes, J. M.; DesMarais, D. J.; Cady, S. L.; Hope, J. M.; Summons, R. E.

    2001-01-01

    This paper describes a study of lipid biomarker composition and carbon isotopic fractionation in cultured Aquificales and natural analogues from Yellowstone National Park. Additional information is contained in the original extended abstract.

  19. Lipid droplet pattern and nondroplet-like structure in two fat mutants of Caenorhabditis elegans revealed by coherent anti-Stokes Raman scattering microscopy.

    PubMed

    Yi, Yung-Hsiang; Chien, Cheng-Hao; Chen, Wei-Wen; Ma, Tian-Hsiang; Liu, Kuan-Yu; Chang, Yu-Sun; Chang, Ta-Chau; Lo, Szecheng J

    2014-01-01

    Lipid is an important energy source and essential component for plasma and organelle membranes in all kinds of cells. Coherent anti-Stokes Raman scattering (CARS) microscopy is a label-free and nonlinear optical technique that can be used to monitor the lipid distribution in live organisms. Here, we utilize CARS microscopy to investigate the pattern of lipid droplets in two live Caenorhabditis elegans mutants (fat-2 and fat-3). The CARS images showed a striking decrease in the size, number, and content of lipid droplets in the fat-2 mutant but a slight difference in the fat-3 mutant as compared with the wild-type worm. Moreover, a nondroplet-like structure with enhanced CARS signal was detected for the first time in the uterus of fat-2 and fat-3 mutants. In addition, transgenic fat-2 mutant expressing a GFP fusion protein of vitellogenin-2 (a yolk lipoprotein) revealed that the enhanced CARS signal colocalized with the GFP signal, which suggests that the nondroplet-like structure is primarily due to the accumulation of yolk lipoproteins. Together, this study implies that CARS microscopy is a potential tool to study the distribution of yolk lipoproteins, in addition to lipid droplets, in live animals. PMID:23979461

  20. Specific polyunsaturated fatty acids modulate lipid delivery and oocyte development in C. elegans revealed by molecular-selective label-free imaging

    PubMed Central

    Chen, Wei-Wen; Yi, Yung-Hsiang; Chien, Cheng-Hao; Hsiung, Kuei-Ching; Ma, Tian-Hsiang; Lin, Yi-Chun; Lo, Szecheng J.; Chang, Ta-Chau

    2016-01-01

    Polyunsaturated fatty acids (PUFAs) exhibit critical functions in biological systems and their importance during animal oocyte maturation has been increasingly recognized. However, the detailed mechanism of lipid transportation for oocyte development remains largely unknown. In this study, the transportation of yolk lipoprotein (lipid carrier) and the rate of lipid delivery into oocytes in live C. elegans were examined for the first time by using coherent anti-Stokes Raman scattering (CARS) microscopy. The accumulation of secreted yolk lipoprotein in the pseudocoelom of live C. elegans can be detected by CARS microscopy at both protein (~1665 cm−1) and lipid (~2845 cm−1) Raman bands. In addition, an image analysis protocol was established to quantitatively measure the levels of secreted yolk lipoprotein aberrantly accumulated in PUFA-deficient fat mutants (fat-1, fat-2, fat-3, fat-4) and PUFA-supplemented fat-2 worms (the PUFA add-back experiments). Our results revealed that the omega-6 PUFAs, not omega-3 PUFAs, play a critical role in modulating lipid/yolk level in the oocytes and regulating reproductive efficiency of C. elegans. This work demonstrates the value of using CARS microscopy as a molecular-selective label-free imaging technique for the study of PUFA regulation and oocyte development in C. elegans. PMID:27535493

  1. Specific polyunsaturated fatty acids modulate lipid delivery and oocyte development in C. elegans revealed by molecular-selective label-free imaging.

    PubMed

    Chen, Wei-Wen; Yi, Yung-Hsiang; Chien, Cheng-Hao; Hsiung, Kuei-Ching; Ma, Tian-Hsiang; Lin, Yi-Chun; Lo, Szecheng J; Chang, Ta-Chau

    2016-01-01

    Polyunsaturated fatty acids (PUFAs) exhibit critical functions in biological systems and their importance during animal oocyte maturation has been increasingly recognized. However, the detailed mechanism of lipid transportation for oocyte development remains largely unknown. In this study, the transportation of yolk lipoprotein (lipid carrier) and the rate of lipid delivery into oocytes in live C. elegans were examined for the first time by using coherent anti-Stokes Raman scattering (CARS) microscopy. The accumulation of secreted yolk lipoprotein in the pseudocoelom of live C. elegans can be detected by CARS microscopy at both protein (~1665 cm(-1)) and lipid (~2845 cm(-1)) Raman bands. In addition, an image analysis protocol was established to quantitatively measure the levels of secreted yolk lipoprotein aberrantly accumulated in PUFA-deficient fat mutants (fat-1, fat-2, fat-3, fat-4) and PUFA-supplemented fat-2 worms (the PUFA add-back experiments). Our results revealed that the omega-6 PUFAs, not omega-3 PUFAs, play a critical role in modulating lipid/yolk level in the oocytes and regulating reproductive efficiency of C. elegans. This work demonstrates the value of using CARS microscopy as a molecular-selective label-free imaging technique for the study of PUFA regulation and oocyte development in C. elegans. PMID:27535493

  2. Lipid droplet pattern and nondroplet-like structure in two fat mutants of Caenorhabditis elegans revealed by coherent anti-Stokes Raman scattering microscopy

    NASA Astrophysics Data System (ADS)

    Yi, Yung-Hsiang; Chien, Cheng-Hao; Chen, Wei-Wen; Ma, Tian-Hsiang; Liu, Kuan-Yu; Chang, Yu-Sun; Chang, Ta-Chau; Lo, Szecheng J.

    2014-01-01

    Lipid is an important energy source and essential component for plasma and organelle membranes in all kinds of cells. Coherent anti-Stokes Raman scattering (CARS) microscopy is a label-free and nonlinear optical technique that can be used to monitor the lipid distribution in live organisms. Here, we utilize CARS microscopy to investigate the pattern of lipid droplets in two live Caenorhabditis elegans mutants (fat-2 and fat-3). The CARS images showed a striking decrease in the size, number, and content of lipid droplets in the fat-2 mutant but a slight difference in the fat-3 mutant as compared with the wild-type worm. Moreover, a nondroplet-like structure with enhanced CARS signal was detected for the first time in the uterus of fat-2 and fat-3 mutants. In addition, transgenic fat-2 mutant expressing a GFP fusion protein of vitellogenin-2 (a yolk lipoprotein) revealed that the enhanced CARS signal colocalized with the GFP signal, which suggests that the nondroplet-like structure is primarily due to the accumulation of yolk lipoproteins. Together, this study implies that CARS microscopy is a potential tool to study the distribution of yolk lipoproteins, in addition to lipid droplets, in live animals.

  3. Genome-Wide Association Analysis for Blood Lipid Traits Measured in Three Pig Populations Reveals a Substantial Level of Genetic Heterogeneity

    PubMed Central

    Yang, Hui; Huang, Xiaochang; Zeng, Zhijun; Zhang, Wanchang; Liu, Chenlong; Fang, Shaoming; Huang, Lusheng; Chen, Congying

    2015-01-01

    Serum lipids are associated with myocardial infarction and cardiovascular disease in humans. Here we dissected the genetic architecture of blood lipid traits by applying genome-wide association studies (GWAS) in 1,256 pigs from Laiwu, Erhualian and Duroc × (Landrace × Yorkshire) populations, and a meta-analysis of GWAS in more than 2,400 pigs from five diverse populations. A total of 22 genomic loci surpassing the suggestive significance level were detected on 11 pig chromosomes (SSC) for six blood lipid traits. Meta-analysis of GWAS identified 5 novel loci associated with blood lipid traits. Comparison of GWAS loci across the tested populations revealed a substantial level of genetic heterogeneity for porcine blood lipid levels. We further evaluated the causality of nine polymorphisms nearby or within the APOB gene on SSC3 for serum LDL-C and TC levels. Of the 9 polymorphisms, an indel showed the most significant association with LDL-C and TC in Laiwu pigs. But the significant association was not identified in the White Duroc × Erhualian F2 resource population, in which the QTL for LDL-C and TC was also detected on SSC3. This indicates that population-specific signals may exist for the SSC3 QTL. Further investigations are warranted to validate this assumption. PMID:26121138

  4. 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.

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

    PubMed

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

    2016-01-01

    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. PMID:27113279

  6. Peptide-induced Asymmetric Distribution of Charged Lipids in a Vesicle Bilayer Revealed by Small-Angle Neutron Scattering

    NASA Astrophysics Data System (ADS)

    Heller, William; Qian, Shuo

    2012-02-01

    Cellular membranes are complex mixtures of lipids, proteins and other small molecules that provide functional, dynamic barriers between the cell and its environment, as well as between environments within the cell. The lipid composition of the membrane is highly specific and controlled in terms of both content and lipid localization. Here, small-angle neutron scattering and selective deuterium labeling were used to probe the impact of the membrane-active peptides melittin and alamethicin on the structure of lipid bilayers composed of a mixture of the lipids dimyristoyl phosphatidylglycerol (DMPG) and chain-perdeuterated dimyristoyl phosphatidylcholine (DMPC). We found that both peptides enriched the outer leaflet of the bilayer with the negatively charged DMPG, creating an asymmetric distribution of lipids. The level of enrichment is peptide concentration-dependent and is stronger for melittin than alamethicin. The enrichment between the inner and outer bilayer leaflets occurs at very low peptide concentrations, and increases with peptide concentration, including when the peptide adopts a membrane-spanning, pore-forming state.

  7. Proteomics to reveal metabolic network shifts towards lipid accumulation following nitrogen deprivation in the diatom Phaeodactylum tricornutum.

    PubMed

    Yang, Zhi-Kai; Ma, Yu-Han; Zheng, Jian-Wei; Yang, Wei-Dong; Liu, Jie-Sheng; Li, Hong-Ye

    2014-01-01

    The marine diatom Phaeodactylum tricornutum is attracting considerable interest as a candidate for biofuel production due to its fast growth and high lipid content. Nitrogen deficiency can increase the lipid content in certain microalgae species, including P. tricornutum. However, the molecular basis of such changes remains unclear without analyzing metabolism at the proteomic level. We attempted to systematically analyze protein expression level changes of P. tricornutum upon N deprivation. We observed translational level changes that could overall redirect the metabolic network from carbon flux towards lipid accumulation. N deprivation led to an increase in the expression of genes involved in nitrogen assimilation and fatty acid biosynthesis and a concomitant decrease in photosynthesis and lipid catabolism enzymes. These molecular level changes are consistent with the observed physiological changes, e.g., in photosynthesis rate and saturated lipid content. Our results provide information at the proteomic level of the key enzymes involved in carbon flux towards lipid accumulation in P. tricornutum and suggest candidates for genetic manipulation in microalgae breeding for biodiesel production. PMID:24600163

  8. Thermoresponsive diblock glycopolymer by RAFT polymerization for lectin recognition.

    PubMed

    Sun, Kan; Xu, Muru; Zhou, Kaichun; Nie, Huali; Quan, Jing; Zhu, Limin

    2016-11-01

    A thermoresponsive double-hydrophilic diblock glycopolymer, poly(diethyl- eneglycol methacrylate)-block-poly(6-O-vinyladipoyl-d-glucose) (PDEGMA-b-POVAG), was successfully prepared by a combination of enzymatic synthesis and reversible addition-fragment chain transfer (RAFT) polymerization protocols using poly(diethyl- eneglycol methacrylate) (PDEGMA) as macro-RAFT agent. The block glycopolymer was characterized by (1)H NMR and GPC. UV-vis, DLS and TEM studies revealed that the glycopolymer PDEGMA-b-POVAG was thermoresponsive with LCST at 31.0°C, and was able to self-assemble into spherical micelles of various sizes in aqueous solution. The glucose pendants in the glycopolymer could interact with the lectin Concanavalin A (Con A), the average hydrodynamic diameters of glycopolymer micelles increased to 170nm from 110nm after recognizing Con A. The diblock glycopolymer micelles have excellent biocompatibility with pig iliac endothelial cells, as measured using the MTT assay, but micelles loaded with Con A could be used to induce apoptosis in human hepatoma SMMC-7721 cells. PMID:27524009

  9. Elasticity of interfacial rafts of hard particles with soft shells.

    PubMed

    Knoche, Sebastian; Kierfeld, Jan

    2015-05-19

    We study an elasticity model for compressed protein monolayers or particle rafts at a liquid interface. Based on the microscopic view of hard-core particles with soft shells, a bead-spring model is formulated and analyzed in terms of continuum elasticity theory. The theory can be applied, for example, to hydrophobin-coated air-water interfaces or, more generally, to liquid interfaces coated with an adsorbed monolayer of interacting hard-core particles. We derive constitutive relations for such particle rafts and describe the buckling of compressed planar liquid interfaces as well as their apparent Poisson ratio. We also use the constitutive relations to obtain shape equations for pendant or buoyant capsules attached to a capillary, and to compute deflated shapes of such capsules. A comparison with capsules obeying the usual Hookean elasticity (without hard cores) reveals that the hard cores trigger capsule wrinkling. Furthermore, it is shown that a shape analysis of deflated capsules with hard-core/soft-shell elasticity gives apparent elastic moduli which can be much higher than the original values if Hookean elasticity is assumed. PMID:25901364

  10. Dynamics and shape of large fire ant rafts

    PubMed Central

    Mlot, Nathan J.; Tovey, Craig; Hu, David L.

    2012-01-01

    To survive floods, fire ants link their bodies together to build waterproof rafts. Such rafts can be quite large, exceeding 100,000 individuals in size. In this study, we make two improvements on a previously reported model on the construction rate of rafts numbering between 3,000 and 10,000 individuals. That model was based upon experimental observations of randomly-directed linear ant trajectories atop the raft. Here, we report anomalous behavior of ants atop larger rafts of up to 23,000 ants. As rafts increase in size, the behavior of ants approaches diffusion, which is in closer alignment with other studies on the foraging and scouting patterns of ants. We incorporate this ant behavior into the model. Our modified model predicts more accurately the growth of large rafts. Our previous model also relied on an assumption of raft circularity. We show that this assumption is not necessary for large rafts, because it follows from the random directionality of the ant trajectories. Our predicted relationship between raft size and circularity closely fits experimental data. PMID:23336030

  11. Revealing the mechanism of passive transport in lipid bilayers via phonon-mediated nanometre-scale density fluctuations

    PubMed Central

    Zhernenkov, Mikhail; Bolmatov, Dima; Soloviov, Dmitry; Zhernenkov, Kirill; Toperverg, Boris P.; Cunsolo, Alessandro; Bosak, Alexey; Cai, Yong Q.

    2016-01-01

    The passive transport of molecules through a cell membrane relies on thermal motions of the lipids. However, the nature of transmembrane transport and the precise mechanism remain elusive and call for a comprehensive study of phonon excitations. Here we report a high resolution inelastic X-ray scattering study of the in-plane phonon excitations in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine above and below the main transition temperature. In the gel phase, for the first time, we observe low-frequency transverse modes, which exhibit a phonon gap when the lipid transitions into the fluid phase. We argue that the phonon gap signifies the formation of short-lived nanometre-scale lipid clusters and transient pores, which facilitate the passive molecular transport across the bilayer plane. Our findings suggest that the phononic motion of the hydrocarbon tails provides an effective mechanism of passive transport, and illustrate the importance of the collective dynamics of biomembranes. PMID:27175859

  12. Revealing the mechanism of passive transport in lipid bilayers via phonon-mediated nanometre-scale density fluctuations

    NASA Astrophysics Data System (ADS)

    Zhernenkov, Mikhail; Bolmatov, Dima; Soloviov, Dmitry; Zhernenkov, Kirill; Toperverg, Boris P.; Cunsolo, Alessandro; Bosak, Alexey; Cai, Yong Q.

    2016-05-01

    The passive transport of molecules through a cell membrane relies on thermal motions of the lipids. However, the nature of transmembrane transport and the precise mechanism remain elusive and call for a comprehensive study of phonon excitations. Here we report a high resolution inelastic X-ray scattering study of the in-plane phonon excitations in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine above and below the main transition temperature. In the gel phase, for the first time, we observe low-frequency transverse modes, which exhibit a phonon gap when the lipid transitions into the fluid phase. We argue that the phonon gap signifies the formation of short-lived nanometre-scale lipid clusters and transient pores, which facilitate the passive molecular transport across the bilayer plane. Our findings suggest that the phononic motion of the hydrocarbon tails provides an effective mechanism of passive transport, and illustrate the importance of the collective dynamics of biomembranes.

  13. Revealing the mechanism of passive transport in lipid bilayers via phonon-mediated nanometre-scale density fluctuations.

    PubMed

    Zhernenkov, Mikhail; Bolmatov, Dima; Soloviov, Dmitry; Zhernenkov, Kirill; Toperverg, Boris P; Cunsolo, Alessandro; Bosak, Alexey; Cai, Yong Q

    2016-01-01

    The passive transport of molecules through a cell membrane relies on thermal motions of the lipids. However, the nature of transmembrane transport and the precise mechanism remain elusive and call for a comprehensive study of phonon excitations. Here we report a high resolution inelastic X-ray scattering study of the in-plane phonon excitations in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine above and below the main transition temperature. In the gel phase, for the first time, we observe low-frequency transverse modes, which exhibit a phonon gap when the lipid transitions into the fluid phase. We argue that the phonon gap signifies the formation of short-lived nanometre-scale lipid clusters and transient pores, which facilitate the passive molecular transport across the bilayer plane. Our findings suggest that the phononic motion of the hydrocarbon tails provides an effective mechanism of passive transport, and illustrate the importance of the collective dynamics of biomembranes. PMID:27175859

  14. Proteomic and Lipidomic Analysis of Nanoparticle Corona upon Contact with Lung Surfactant Reveals Differences in Protein, but Not Lipid Composition.

    PubMed

    Raesch, Simon Sebastian; Tenzer, Stefan; Storck, Wiebke; Rurainski, Alexander; Selzer, Dominik; Ruge, Christian Arnold; Perez-Gil, Jesus; Schaefer, Ulrich Friedrich; Lehr, Claus-Michael

    2015-12-22

    Pulmonary surfactant (PS) constitutes the first line of host defense in the deep lung. Because of its high content of phospholipids and surfactant specific proteins, the interaction of inhaled nanoparticles (NPs) with the pulmonary surfactant layer is likely to form a corona that is different to the one formed in plasma. Here we present a detailed lipidomic and proteomic analysis of NP corona formation using native porcine surfactant as a model. We analyzed the adsorbed biomolecules in the corona of three NP with different surface properties (PEG-, PLGA-, and Lipid-NP) after incubation with native porcine surfactant. Using label-free shotgun analysis for protein and LC-MS for lipid analysis, we quantitatively determined the corona composition. Our results show a conserved lipid composition in the coronas of all investigated NPs regardless of their surface properties, with only hydrophilic PEG-NPs adsorbing fewer lipids in total. In contrast, the analyzed NP displayed a marked difference in the protein corona, consisting of up to 417 different proteins. Among the proteins showing significant differences between the NP coronas, there was a striking prevalence of molecules with a notoriously high lipid and surface binding, such as, e.g., SP-A, SP-D, DMBT1. Our data indicate that the selective adsorption of proteins mediates the relatively similar lipid pattern in the coronas of different NPs. On the basis of our lipidomic and proteomic analysis, we provide a detailed set of quantitative data on the composition of the surfactant corona formed upon NP inhalation, which is unique and markedly different to the plasma corona. PMID:26575243

  15. Raft River wellfield testing and analysis

    SciTech Connect

    Russell, B.F.

    1982-04-01

    The testing procedures and an overview of the expected performance of the Raft River wellfield during plant operation are presented. Four well-testing procedures were used to evaluate the seven geothermal wells: (1) artesian flow and airlift tests during and shortly after drilling; (2) short duration constant rate and variable-head artesian flow tests following drilling; (3) a series of pulse discharge and injection tests of short duration, with constant rate and variable head; and (4) pumping and injection tests of up to 30 days duration using permanently installed pumps. Productivity curves were plotted for each of the exploratory and production wells. It was concluded that the Raft River wellfield has the capability of supplying the necessary flow to operate the 5MW(e) facility. (MJF)

  16. Remorin, a Solanaceae Protein Resident in Membrane Rafts and Plasmodesmata, Impairs Potato virus X Movement[W

    PubMed Central

    Raffaele, Sylvain; Bayer, Emmanuelle; Lafarge, David; Cluzet, Stéphanie; German Retana, Sylvie; Boubekeur, Tamy; Leborgne-Castel, Nathalie; Carde, Jean-Pierre; Lherminier, Jeannine; Noirot, Elodie; Satiat-Jeunemaître, Béatrice; Laroche-Traineau, Jeanny; Moreau, Patrick; Ott, Thomas; Maule, Andrew J.; Reymond, Philippe; Simon-Plas, Françoise; Farmer, Edward E.; Bessoule, Jean-Jacques; Mongrand, Sébastien

    2009-01-01

    Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in ∼70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane. PMID:19470590

  17. Transcription analysis of genes involved in lipid metabolism reveals the role of chromium in reducing body fat in animal models.

    PubMed

    Sadeghi, Mostafa; Najaf Panah, Mohammad Javad; Bakhtiarizadeh, Mohammad Reza; Emami, Ali

    2015-10-01

    Chromium was proposed to be an essential trace element over 50 years ago and has been accepted as an essential element for over 30 years. The recent studies indicated that the addition of supra nutritional amounts of chromium to the diet can only be considered as having pharmacological effects. However, the precise mechanism through which chromium acts on lipid, carbohydrate, protein and nucleic acid metabolism are relatively poor studied. To uncover, at least partially, the role of chromium in lipid metabolism, in this study, we evaluated the expression status of eight important genes, involved in fat biosynthesis and lipid metabolism, in four different tissue types (liver, subcutaneous fat, visceral fat, and longissimus muscle) in domestic goat kids feeding on three different chromium levels. The quantitative real-time PCR (RT-PCR) was established for expression analyses with HSP90 gene was used as reference gene. The results showed that supplementation of goats with 1.5mg/day chromium significantly decreases the expression of the ACC1, DGAT1, FABP4, FAS, HSL, LEP genes, but does not affect the expression of the LPL and SCD1 genes in all studied tissues. This study highlights, for the first time, the role of supra nutritional levels of chromium in lipid biosynthesis and metabolism. These findings are of especial importance for improving meat quality in domestic animals. PMID:26302911

  18. Multimerization of Human Immunodeficiency Virus Type 1 Gag Promotes Its Localization to Barges, Raft-Like Membrane Microdomains

    PubMed Central

    Lindwasser, O. Wolf; Resh, Marilyn D.

    2001-01-01

    The Gag polyprotein of human immunodeficiency virus type 1 (HIV-1) organizes the assembly of nascent virions at the plasma membrane of infected cells. Here we demonstrate that a population of Gag is present in distinct raft-like membrane microdomains that we have termed “barges.” Barges have a higher density than standard rafts, most likely due to the presence of oligomeric Gag-Gag assembly complexes. The regions of the Gag protein responsible for barge targeting were mapped by examining the flotation behavior of wild-type and mutant proteins on Optiprep density gradients. N-myristoylation of Gag was necessary for association with barges. Removal of the NC and p6 domains shifted much of the Gag from barges into typical raft fractions. These data are consistent with a model in which multimerization of myristoylated Gag proteins drives association of Gag oligomers into raft-like barges. The functional significance of barge association was revealed by several lines of evidence. First, Gag isolated from virus-like particles was almost entirely localized in barges. Moreover, a comparison of wild-type Gag with Fyn(10)Gag, a chimeric protein containing the N-terminal sequence of Fyn, revealed that Fyn(10)Gag exhibited increased affinity for barges and a two- to fourfold increase in particle production. These results imply that association of Gag with raft-like barge membrane microdomains plays an important role in the HIV-1 assembly process. PMID:11483736

  19. HIV gp41-Mediated Membrane Fusion Occurs at Edges of Cholesterol-Rich Lipid Domains

    PubMed Central

    Yang, Sung-Tae; Kiessling, Volker; Simmons, James A.; White, Judith M.; Tamm, Lukas K.

    2015-01-01

    Lipid rafts in plasma membranes have emerged as possible platforms for entry of HIV and other viruses into cells. However, how lipid phase heterogeneity contributes to viral entry is little known due to the fine-grained and still poorly understood complexity of biological membranes. We used model systems mimicking HIV envelopes and T-cell membranes and showed that raft-like (Lo phase) lipid domains are necessary and sufficient for efficient membrane targeting and fusion. Interestingly, membrane binding and fusion was low in homogeneous Ld and Lo phase membranes, indicating that lipid phase heterogeneity is essential. The HIV fusion peptide preferentially targeted to Lo/Ld boundary regions and promoted full fusion at the interface between ordered and disordered lipids. Ld phase vesicles proceeded only to hemifusion. Thus, we propose that the edges, but not the areas of raft-like ordered lipid domains are vital for HIV entry and membrane fusion. PMID:25915200

  20. Planning and execution of Raft River stimulation treatments

    SciTech Connect

    Verity, R.V.; Crichlow, H.B.

    1980-02-07

    The following topics are discussed for two Raft River Valley wells: well characteristics and treatment objectives, treatment selection and design, treatment history, mechanical arrangements and job costs. (MHR)

  1. 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

  2. Cholesterol's location in lipid bilayers.

    PubMed

    Marquardt, Drew; Kučerka, Norbert; Wassall, Stephen R; Harroun, Thad A; Katsaras, John

    2016-09-01

    It is well known that cholesterol modifies the physical properties of lipid bilayers. For example, the much studied liquid-ordered Lo phase contains rapidly diffusing lipids with their acyl chains in the all trans configuration, similar to gel phase bilayers. Moreover, the Lo 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 is in the vicinity of the lipid-water interface. In this article 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. PMID:27056099

  3. iTRAQ-based proteomic analysis of plasma reveals abnormalities in lipid metabolism proteins in chronic kidney disease-related atherosclerosis.

    PubMed

    Luczak, Magdalena; Formanowicz, Dorota; Marczak, Łukasz; Suszyńska-Zajczyk, Joanna; Pawliczak, Elżbieta; Wanic-Kossowska, Maria; Stobiecki, Maciej

    2016-01-01

    Patients with chronic kidney disease (CKD) have a considerably higher risk of death due to cardiovascular causes. Using an iTRAQ MS/MS approach, we investigated the alterations in plasma protein accumulation in patients with CKD and classical cardiovascular disease (CVD) without CKD. The proteomic analysis led to the identification of 130 differentially expressed proteins among CVD and CKD patients and healthy volunteers. Bioinformatics analysis revealed that 29 differentially expressed proteins were involved in lipid metabolism and atherosclerosis, 20 of which were apolipoproteins and constituents of high-density lipoprotein (HDL) and low-density lipoprotein (LDL). Although dyslipidemia is common in CKD patients, we found that significant changes in apolipoproteins were not strictly associated with changes in plasma lipid levels. A lack of correlation between apoB and LDL concentration and an inverse relationship of some proteins with the HDL level were revealed. An increased level of apolipoprotein AIV, adiponectin, or apolipoprotein C, despite their anti-atherogenic properties, was not associated with a decrease in cardiovascular event risk in CKD patients. The presence of the distinctive pattern of apolipoproteins demonstrated in this study may suggest that lipid abnormalities in CKD are characterized by more qualitative abnormalities and may be related to HDL function rather than HDL deficiency. PMID:27600335

  4. iTRAQ-based proteomic analysis of plasma reveals abnormalities in lipid metabolism proteins in chronic kidney disease-related atherosclerosis

    PubMed Central

    Luczak, Magdalena; Formanowicz, Dorota; Marczak, Łukasz; Suszyńska-Zajczyk, Joanna; Pawliczak, Elżbieta; Wanic-Kossowska, Maria; Stobiecki, Maciej

    2016-01-01

    Patients with chronic kidney disease (CKD) have a considerably higher risk of death due to cardiovascular causes. Using an iTRAQ MS/MS approach, we investigated the alterations in plasma protein accumulation in patients with CKD and classical cardiovascular disease (CVD) without CKD. The proteomic analysis led to the identification of 130 differentially expressed proteins among CVD and CKD patients and healthy volunteers. Bioinformatics analysis revealed that 29 differentially expressed proteins were involved in lipid metabolism and atherosclerosis, 20 of which were apolipoproteins and constituents of high-density lipoprotein (HDL) and low-density lipoprotein (LDL). Although dyslipidemia is common in CKD patients, we found that significant changes in apolipoproteins were not strictly associated with changes in plasma lipid levels. A lack of correlation between apoB and LDL concentration and an inverse relationship of some proteins with the HDL level were revealed. An increased level of apolipoprotein AIV, adiponectin, or apolipoprotein C, despite their anti-atherogenic properties, was not associated with a decrease in cardiovascular event risk in CKD patients. The presence of the distinctive pattern of apolipoproteins demonstrated in this study may suggest that lipid abnormalities in CKD are characterized by more qualitative abnormalities and may be related to HDL function rather than HDL deficiency. PMID:27600335

  5. Brucella abortus 16S rRNA and lipid A reveal a phylogenetic relationship with members of the alpha-2 subdivision of the class Proteobacteria.

    PubMed Central

    Moreno, E; Stackebrandt, E; Dorsch, M; Wolters, J; Busch, M; Mayer, H

    1990-01-01

    On the basis of ribosomal 16S sequence comparison, Brucella abortus has been found to be a member of the alpha-2 subdivision of the class Proteobacteria (formerly named purple photosynthetic bacteria and their nonphototrophic relatives). Within the alpha-2 subgroup, brucellae are specifically related to rickettsiae, agrobacteria, and rhizobiae, organisms that also have the faculty or the obligation of living in close association to eucaryotic cells. The composition of Brucella lipid A suggests a close phylogenetical relationship with members of the alpha-2 group. The chemical analysis of the lipid A fraction revealed that Brucella species contain both glucosamine and diaminoglucose, thus suggesting the presence of a so-called mixed lipid A type. The serological analysis with polyclonal and monoclonal antibodies is in agreement with the existence of mixed lipid A type in B. abortus. The amide-linked fatty acid present as acyl-oxyacyl residues were 3-O-C(16:0)12:0, 3-O-C(16:0)13:0, 3-O-C(16:0)14:0, and 3-O-C(18:0)14:0. The only amide-linked unsubstituted fatty acid detected was 3-OH-C16:0. The ester-linked fatty acids are 3-OH-C16:0, 3-OH-C18:0, C16:0, C17:0, and C18:0. Significant amounts of the large-chain 27-OH-C28:0 were detected together with traces of 25-OH-C26:0 and 29-OH-C30:0. Comparison of the Brucella lipid composition with that of the other Proteobacteria also suggests a close phylogenetical relationship with members of the alpha-2 subdivision. The genealogical grouping of Brucella species with pericellular and intracellular plant and animal pathogens as well as with intracellular plant symbionts suggests a possible evolution of Brucella species from plant-arthropod-associated bacteria. PMID:2113907

  6. 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

  7. Lipid membrane domains in cell surface and vacuolar systems.

    PubMed

    Kobayashi, T; Hirabayashi, Y

    2000-01-01

    Detergent insoluble sphingolipid-cholesterol enriched 'raft'-like membrane microdomains have been implicated in a variety of biological processes including sorting, trafficking, and signaling. Mutant cells and knockout animals of sphingolipid biosynthesis are clearly useful to understand the biological roles of lipid components in raft-like domains. It is suggested that raft-like domains distribute in internal vacuolar membranes as well as plasma membranes. In addition to sphingolipid-cholesterol-rich membrane domains, recent studies suggest the existence of another lipid-membrane domain in the endocytic pathway. This domain is enriched with a unique phospholipid, lysobisphosphatidic acid (LBPA) and localized in the internal membrane of multivesicular endosome. LBPA-rich membrane domains are involved in lipid and protein sorting within the endosomal system. Possible interaction between sphingolipids and LBPA in sphingolipid-storage disease is discussed. PMID:11201787

  8. Composition of Hydrothermal Vent Microbial Communities as Revealed by Analyses of Signature Lipids, Stable Carbon Isotopes and Aquificales Cultures

    NASA Technical Reports Server (NTRS)

    Jahnke, Linda L.; Eder, Wolfgang; Huber, Robert; Hinrichs, Kai-Uwe; Hayes, John M.; Cady, Sherry L.; DesMarais, David J.; Hope, Janet M.; Summons, Roger E.

    2001-01-01

    Extremely thermophilic microbial communities associated with the siliceous vent walls and outflow channel of Octopus Spring, Yellowstone National Park, have been examined for lipid biomarker and carbon isotopic signatures. These data were compared with that obtained from representatives of three Aquificales genera. Thermocrinis ruber, Thermocrinis sp. HI, Hydrogenobacter thermophilus, Aquifex pyrophilus and Aquifex aeolicus all contained phospholipids composed not only of the usual ester-linked fatty acids, but also ether-linked alkyl moieties. The fatty acids of all cultured organisms were dominated by very distinct pattern of n-C-20:1 and cy-C-21 compounds. The alkyl glycerol ethers were present primarily as C-18:0 monoethers with the exception of the Aquifex spp. in which dialkyl glycerol ethers with a boarder carbon-number distribution were also present. These Aquificales biomarker lipids were the major constituents in the lipid extracts of the Octopus Spring microbial samples. Two natural samples, a microbial biofilm growing in association with deposition of amorphous silica on the vent walls at 92 C, and the well-known "pink-streamer community" (PSC), siliceous filaments of a microbial consortia growing in the outflow channel at 87 C were analyzed. Both the biofilm and PSC samples contained mono- and dialkyl glycerol ethers with a prevalence of C-18 and C-20 alkyls. Phospholipid fatty acids were comprised of both the characteristic. Additional information is contained in the original extended abstract.

  9. Lipid Membrane Deformation Accompanied by Disk-to-Ring Shape Transition of Cholesterol-Rich Domains.

    PubMed

    Ryu, Yong-Sang; Yoo, Daehan; Wittenberg, Nathan J; Jordan, Luke R; Lee, Sin-Doo; Parikh, Atul N; Oh, Sang-Hyun

    2015-07-15

    During vesicle budding or endocytosis, biomembranes undergo a series of lipid- and protein-mediated deformations involving cholesterol-enriched lipid rafts. If lipid rafts of high bending rigidities become confined to the incipient curved membrane topology such as a bud-neck interface, they can be expected to reform as ring-shaped rafts. Here, we report on the observation of a disk-to-ring shape morpho-chemical transition of a model membrane in the absence of geometric constraints. The raft shape transition is triggered by lateral compositional heterogeneity and is accompanied by membrane deformation in the vertical direction, which is detected by height-sensitive fluorescence interference contrast microscopy. Our results suggest that a flat membrane can become curved simply by dynamic changes in local chemical composition and shape transformation of cholesterol-rich domains. PMID:26053547

  10. The ether lipid-deficient mouse: tracking down plasmalogen functions.

    PubMed

    Gorgas, Karin; Teigler, Andre; Komljenovic, Dorde; Just, Wilhelm W

    2006-12-01

    Chemical and physico-chemical properties as well as physiological functions of major mammalian ether-linked glycerolipids, including plasmalogens were reviewed. Their chemical structures were described and their effect on membrane fluidity and membrane fusion discussed. The recent generation of mouse models with ether lipid deficiency offered the possibility to study ether lipid and particularly plasmalogen functions in vivo. Ether lipid-deficient mice revealed severe phenotypic alterations, including arrest of spermatogenesis, development of cataract and defects in central nervous system myelination. In several cell culture systems lack of plasmalogens impaired intracellular cholesterol distribution affecting plasma membrane functions and structural changes of ER and Golgi cisternae. Based on these phenotypic anomalies that were accurately described conclusions were drawn on putative functions of plasmalogens. These functions were related to cell-cell or cell-extracellular matrix interactions, formation of lipid raft microdomains and intracellular cholesterol homeostasis. There are several human disorders, such as Zellweger syndrome, rhizomelic chondrodysplasia punctata, Alzheimer's disease, Down syndrome, and Niemann-Pick type C disease that are distinguished by altered tissue plasmalogen concentrations. The role plasmalogens might play in the pathology of these disorders is discussed. PMID:17027098

  11. 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. PMID:23770033

  12. Selective Uptake of a Fructose Glycopolymer Prepared by RAFT Polymerization into Human Breast Cancer Cells.

    PubMed

    von der Ehe, Christian; Rinkenauer, Alexandra; Weber, Christine; Szamosvari, David; Gottschaldt, Michael; Schubert, Ulrich S

    2016-04-01

    A new methacrylic fructose glycomonomer is synthesized and copolymerized with N-isopropyl acrylamide by reversible addition fragmentation chain transfer (RAFT) poly-merization. By additional copolymerization of the analog mannose, glucose, and galactose glycomonomers, a set of glycopolymers is obtained which vary in the type of sugar attached to the polyacrylamide backbone. The glycopolymers are subsequently deprotected and characterized by size exclusion chromatography, FT-IR and NMR spectroscopy, elemental analysis, as well as turbidimetry, revealing the thermoresponsive character of all synthesized glycopolymers. The deprotected glycopolymers are subsequently labeled with a Rhodamine B derivative, utilizing the thiol-functionalities derived from the RAFT endgroups. As concluded from the ArlamaBlue assay, the glycopolymers are not cytotoxic. Finally, cellular uptake studies reveal a higher uptake of the fructose polymer into MDA-MB-231 breast cancer cells compared to the other glycopolymers, which demonstrates the high potential of fructosylated polymers for potential applications in the targeted treatment of breast cancer. PMID:26688011

  13. The proteome of cytosolic lipid droplets isolated from differentiated Caco-2/TC7 enterocytes reveals cell-specific characteristics

    PubMed Central

    Bouchoux, Julien; Beilstein, Frauke; Pauquai, Thomas; Guerrera, I. Chiara; Chateau, Danielle; Ly, Nathalie; Alqub, Malik; Klein, Christophe; Chambaz, Jean; Rousset, Monique; Lacorte, Jean-Marc; Morel, Etienne; Demignot, Sylvie

    2011-01-01

    Background information. Intestinal absorption of alimentary lipids is a complex process ensured by enterocytes and leading to TRL [TAG (triacylglycerol)-rich lipoprotein] assembly and secretion. The accumulation of circulating intestine-derived TRL is associated with atherosclerosis, stressing the importance of the control of postprandial hypertriglyceridaemia. During the postprandial period, TAGs are also transiently stored as CLDs (cytosolic lipid droplets) in enterocytes. As a first step for determining whether CLDs could play a role in the control of enterocyte TRL secretion, we analysed the protein endowment of CLDs isolated by sucrose-gradient centrifugation from differentiated Caco-2/TC7 enterocytes, the only human model able to secrete TRL in culture and to store transiently TAGs as CLDs when supplied with lipids. Cells were analysed after a 24 h incubation with lipid micelles and thus in a state of CLD-associated TAG mobilization. Results. Among the 105 proteins identified in the CLD fraction by LC-MS/MS (liquid chromatography coupled with tandem MS), 27 were directly involved in lipid metabolism pathways potentially relevant to enterocyte-specific functions. The transient feature of CLDs was consistent with the presence of proteins necessary for fatty acid activation (acyl-CoA synthetases) and for TAG hydrolysis. In differentiated Caco-2/TC7 enterocytes, we identified for the first time LPCAT2 (lysophosphatidylcholine acyltransferase 2), involved in PC (phosphatidylcholine) synthesis, and 3BHS1 (3-β-hydroxysteroid dehydrogenase 1), involved in steroid metabolism, and confirmed their partial CLD localization by immunofluorescence. In enterocytes, LPCAT2 may provide an economical source of PC, necessary for membrane synthesis and lipoprotein assembly, from the lysoPC present in the intestinal lumen. We also identified proteins involved in lipoprotein metabolism, such as ApoA-IV (apolipoprotein A-IV), which is specifically expressed by enterocytes and has

  14. Composition of Hydrothermal Vent Microbial Communities as Revealed by Analyses of Signature Lipids, Stable Carbon Isotopes and Aquificales Cultures

    NASA Technical Reports Server (NTRS)

    Jahnke, Linda L.; Edger, Wolfgang; Huber, Robert; Hinrichs, Kai-Uwe; Hayes, John M.; DesMarais, David J.; Cady, Sherry; Hope, Janet M.; Summons, Roger E.; DeVincenzi, Donald L. (Technical Monitor)

    2001-01-01

    Extremely thermophilic microbial communities associated with the siliceous vent walls and outflow channel of Octopus Spring, Yellowstone National Park, have been examined for lipid biomarkers and carbon isotopic signatures. These data were compared with that obtained from representatives of three Aquificales genera. Thermocrinis ruber. "Thermocrinis sp. HI", Hydrogenobacter thermophilus TK-6, Aquifex pyrophilus and Aquifex aeolicus all contained phospholipids composed not only of the usual ester-linked fatty acids, but also ether-linked alkyls. The fatty acids of all cultured organisms were dominated by a very distinct pattern of n-C-20:1 and cy-C-21 compounds. The alkyl glycerol ethers were present primarily as CIS() monoethers with the expection of the Aquifex spp. in which dialkyl glycerol ethers with a boarder carbon-number distribution were also present. These Aquificales biomarker lipids were the major constituents in the lipid extracts of the Octopus Spring microbial samples. Two natural samples, a microbial biofilm growing in association with deposition of amorphous silica on the vent walls at 92 C, and the well-known 'pink-streamers community' (PSC), siliceous filaments of a microbial consortia growing in the upper outflow channel at 87 C were analyzed. Both the biofilm and PSC samples contained mono and dialkyl glycerol ethers with a prevalence of C-18 and C-20 alkyls. Phospholipid fatty acids were comprised of both the characteristic Aquificales n-C-20:1 and cy-C-21, and in addition, a series of iso-branched fatty acids from i-C-15:0 to i-C-21:0, With i-C-17:0 dominant in the PSC and i-C-19:0 in the biofilm, suggesting the presence of two major bacterial groups. Bacteriohopanepolyols were absent and the minute quantities of archaeol detected showed that Archaea were only minor constituents. Carbon isotopic compositions of the PSC yielded information about community structure and likely physiology. Biomass was C-13-depleted (10.9%) relative to available

  15. Interplay of cytoskeletal activity and lipid phase stability in dynamic protein recruitment and clustering.

    PubMed

    Gómez-Llobregat, Jordi; Buceta, Javier; Reigada, Ramon

    2013-01-01

    Recent experiments have revealed that some membrane proteins aggregate to form clusters. This type of process has been proven to be dynamic and to be actively maintained by external kinetics. Additionally, this dynamic recruiting is cholesterol- and actin-dependent, suggesting that raft organization and cytoskeleton rearrangement play a crucial role. In the present study, we propose a simple model that provides a general framework to describe the dynamical behavior of lipid-protein assemblies. Our results suggest that lipid-mediated interactions and cytoskeleton-anchored proteins contribute to the modulation of such behavior. In particular, we find a resonant condition between the membrane protein and cytoskeleton dynamics that results in the invariance of the ratio of clustered proteins that is found in in vivo experimental observations. PMID:24018870

  16. Analysis of lipid-composition changes in plasma membrane microdomains[S

    PubMed Central

    Ogiso, Hideo; Taniguchi, Makoto; Okazaki, Toshiro

    2015-01-01

    Sphingolipids accumulate in plasma membrane microdomain sites, such as caveolae or lipid rafts. Such microdomains are considered to be important nexuses for signal transduction, although changes in the microdomain lipid components brought about by signaling are poorly understood. Here, we applied a cationic colloidal silica bead method to analyze plasma membrane lipids from monolayer cells cultured in a 10 cm dish. The detergent-resistant fraction from the silica bead-coated membrane was analyzed by LC-MS/MS to evaluate the microdomain lipids. This method revealed that glycosphingolipids composed the microdomains as a substitute for sphingomyelin (SM) in mouse embryonic fibroblasts (tMEFs) from an SM synthase 1/2 double KO (DKO) mouse. The rate of formation of the detergent-resistant region was unchanged compared with that of WT-tMEFs. C2-ceramide (Cer) stimulation caused greater elevations in diacylglycerol and phosphatidic acid levels than in Cer levels within the microdomains of WT-tMEFs. We also found that lipid changes in the microdomains of SM-deficient DKO-tMEFs caused by serum stimulation occurred in the same manner as that of WT-tMEFs. This practical method for analyzing membrane lipids will facilitate future comprehensive analyses of membrane microdomain-associated responses. PMID:26116739

  17. Transcriptional profile reveals altered hepatic lipid and cholesterol metabolism in hyposulfatemic NaS1 null mice.

    PubMed

    Dawson, Paul Anthony; Gardiner, Brooke; Grimmond, Sean; Markovich, Daniel

    2006-07-12

    Sulfate plays an essential role in human growth and development, and its circulating levels are maintained by the renal Na+-SO42- cotransporter, NaS1. We previously generated a NaS1 knockout (Nas1-/-) mouse, an animal model for hyposulfatemia, that exhibits reduced growth and liver abnormalities including hepatomegaly. In this study, we investigated the hepatic gene expression profile of Nas1-/- mice using oligonucleotide microarrays. The mRNA expression levels of 92 genes with known functional roles in metabolism, cell signaling, cell defense, immune response, cell structure, transcription, or protein synthesis were increased (n = 51) or decreased (n = 41) in Nas1-/- mice when compared with Nas1+/+ mice. The most upregulated transcript levels in Nas1-/- mice were found for the sulfotransferase genes, Sult3a1 (approximately 500% increase) and Sult2a2 (100% increase), whereas the metallothionein-1 gene, Mt1, was among the most downregulated genes (70% decrease). Several genes involved in lipid and cholesterol metabolism, including Scd1, Acly, Gpam, Elov16, Acsl5, Mvd, Insig1, and Apoa4, were found to be upregulated (> or = 30% increase) in Nas1-/- mice. In addition, Nas1-/- mice exhibited increased levels of hepatic lipid (approximately 16% increase), serum cholesterol (approximately 20% increase), and low-density lipoprotein (approximately 100% increase) and reduced hepatic glycogen (approximately 50% decrease) levels. In conclusion, these data suggest an altered lipid and cholesterol metabolism in the hyposulfatemic Nas1-/- mouse and provide new insights into the metabolic state of the liver in Nas1-/- mice. PMID:16621889

  18. Composition and Localization of Lipids in Penaeus merguiensis Ovaries during the Ovarian Maturation Cycle as Revealed by Imaging Mass Spectrometry

    PubMed Central

    Chansela, Piyachat; Goto-Inoue, Naoko; Zaima, Nobuhiro; Hayasaka, Takahiro; Sroyraya, Morakot; Kornthong, Napamanee; Engsusophon, Attakorn; Tamtin, Montakan; Chaisri, Chatchawalee; Sobhon, Prasert; Setou, Mitsutoshi

    2012-01-01

    Ovary maturation, oocyte differentiation, and embryonic development in shrimp are highly dependent on nutritional lipids taken up by female broodstocks. These lipids are important as energy sources as well as for cell signaling. In this study, we report on the compositions of major lipids, i.e. phosphatidylcholines (PCs), triacylglycerols (TAGs), and fatty acids (FAs), in the ovaries of the banana shrimp, Penaeus merguiensis, during ovarian maturation. Thin-layer chromatography analysis showed that the total PC and TAG signal intensities increased during ovarian maturation. Further, by using gas chromatography, we found that (1) FAs 14∶0, 16∶1, 18∶1, 18∶2, 20∶1, and 22∶6 proportionally increased as ovarian development progressed to more mature stages; (2) FAs 16∶0, 18∶0, 20∶4, and 20∶5 proportionally decreased; and (3) FAs 15∶0, 17∶0, and 20∶2 remained unchanged. By using imaging mass spectrometry, we found that PC 16∶0/16∶1 and TAG 18∶1/18∶2/22∶6 were detected in oocytes stages 1 and 2. PCs 16∶1/20∶4, 16∶0/22∶6, 18∶3/22∶6, 18∶1/22∶6, 20∶5/22∶6, and 22∶6/22∶6 and TAGs 16∶0/16∶1/18∶3, 16∶0/18∶1/18∶3, 16∶0/18∶1/18∶1, and 16∶0/18∶2/22∶6 were present in all stages of oocytes. In contrast, the PC- and TAG-associated FAs 20∶4, 20∶5, and 22∶6 showed high signal intensities in stage 3 and 4 oocytes. These FAs may act as nutrition sources as well as signaling molecules for developing embryos and the hatching process. Knowledge of lipid compositions and localization could be helpful for formulating the diet for female broodstocks to promote fecundity and larval production. PMID:22432001

  19. Raft River aquaculture project. Final report

    SciTech Connect

    Beleau, M.H.; Woiwode, J.G.

    1980-07-01

    The commercial potential for geothermal aquaculture was evaluated for 2 years at the Department of Energy's Raft River geothermal site in southcentral Idaho. Common carp '(Cyprinus carpio) and channel catfish (Ictalurus punctatus) were selected as culture species. Objectives of the study included investigation of: (1) growth rates; (2) nutrition trials; (3) histological and physiological parameters; (4) bioaccumulation of heavy metals; and (5) reproductive capacity. The second year project efforts were primarily studying the effects of geothermal water on the reproductive capacity of common carp by: (1) determining the effects of geothermal water on gonadal development of common carp; and (2) determining the effects of geothermal water on common carp embryogenesis.

  20. 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. PMID:27056046

  1. 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.

  2. Formation of functional cell membrane domains: the interplay of lipid- and protein-mediated interactions.

    PubMed Central

    Harder, Thomas

    2003-01-01

    Numerous cell membrane associated processes, including signal transduction, membrane sorting, protein processing and virus trafficking take place in membrane subdomains. Protein-protein interactions provide the frameworks necessary to generate biologically functional membrane domains. For example, coat proteins define membrane areas destined for sorting processes, viral proteins self-assemble to generate a budding virus, and adapter molecules organize multimolecular signalling assemblies, which catalyse downstream reactions. The concept of raft lipid-based membrane domains provides a different principle for compartmentalization and segregation of membrane constituents. Accordingly, rafts are defined by the physical properties of the lipid bilayer and function by selective partitioning of membrane lipids and proteins into membrane domains of specific phase behaviour and lipid packing. Here, I will discuss the interplay of these independent principles of protein scaffolds and raft lipid microdomains leading to the generation of biologically functional membrane domains. PMID:12803918

  3. 'Green' reversible addition-fragmentation chain-transfer (RAFT) polymerization

    NASA Astrophysics Data System (ADS)

    Semsarilar, Mona; Perrier, Sébastien

    2010-10-01

    Reversible addition-fragmentation chain-transfer (RAFT) polymerization has revolutionized the field of polymer synthesis as a versatile tool for the production of complex polymeric architectures. As for all chemical processes, research and development in RAFT have to focus on the design and application of chemical products and processes that have a minimum environmental impact, and follow the principles of 'green' chemistry. In this Review, we summarize some of the green features of the RAFT process, and review the recent advances in the production of degradable polymers obtained from RAFT polymerization. Its use to modify biodegradable and renewable inorganic and organic materials to yield more functional products with enhanced applications is also covered. RAFT is a promising candidate for answering both the increasing need of modern society to employ highly functional polymeric materials and the global requirements for developing sustainable chemicals and processes.

  4. HR-MAS MRS of the pancreas reveals reduced lipid and elevated lactate and taurine associated with early pancreatic cancer.

    PubMed

    Wang, Alan S; Lodi, Alessia; Rivera, Lee B; Izquierdo-Garcia, Jose L; Firpo, Matthew A; Mulvihill, Sean J; Tempero, Margaret A; Bergers, Gabriele; Ronen, Sabrina M

    2014-11-01

    The prognosis for patients with pancreatic cancer is extremely poor, as evidenced by the disease's five-year survival rate of ~5%. New approaches are therefore urgently needed to improve detection, treatment, and monitoring of pancreatic cancer. MRS-detectable metabolic changes provide useful biomarkers for tumor detection and response-monitoring in other cancers. The goal of this study was to identify MRS-detectable biomarkers of pancreatic cancer that could enhance currently available imaging approaches. We used (1) H high-resolution magic angle spinning MRS to probe metabolite levels in pancreatic tissue samples from mouse models and patients. In mice, the levels of lipids dropped significantly in pancreata with lipopolysaccharide-induced inflammation, in pancreata with pre-cancerous metaplasia (4 week old p48-Cre;LSL-Kras(G12D) mice), and in pancreata with pancreatic intraepithelial neoplasia, which precedes invasive pancreatic cancer (8 week old p48-Cre LSL-Kras(G12D) mice), to 26 ± 19% (p = 0.03), 19 ± 16% (p = 0.04), and 26 ± 10% (p = 0.05) of controls, respectively. Lactate and taurine remained unchanged in inflammation and in pre-cancerous metaplasia but increased significantly in pancreatic intraepithelial neoplasia to 266 ± 61% (p = 0.0001) and 999 ± 174% (p < 0.00001) of controls, respectively. Importantly, analysis of patient biopsies was consistent with the mouse findings. Lipids dropped in pancreatitis and in invasive cancer biopsies to 29 ± 15% (p = 0.01) and 26 ± 38% (p = 0.02) of normal tissue. In addition, lactate and taurine levels remained unchanged in inflammation but rose in tumor samples to 244 ± 155% (p = 0.02) and 188 ± 67% (p = 0.02), respectively, compared with normal tissue. Based on these findings, we propose that a drop in lipid levels could serve to inform on pancreatitis and cancer-associated inflammation, whereas elevated lactate and taurine could serve to identify the presence of pancreatic intraepithelial neoplasia and

  5. Degradable PEGylated Protein Conjugates Utilizing RAFT Polymerization

    PubMed Central

    Decker, Caitlin G.; Maynard, Heather D.

    2015-01-01

    Poly(ethylene glycol) (PEG)-protein therapeutics exhibit enhanced pharmacokinetics, but have drawbacks including decreased protein activities and polymer accumulation in the body. Therefore a major aim for second-generation polymer therapeutics is to introduce degradability into the backbone. Herein we describe the synthesis of poly(poly(ethylene glycol methyl ether methacrylate)) (pPEGMA) degradable polymers with protein-reactive end-groups via reversible addition-fragmentation chain transfer (RAFT) polymerization, and the subsequent covalent attachment to lysozyme through a reducible disulfide linkage. RAFT copolymerization of cyclic ketene acetal (CKA) monomer 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) with PEGMA yielded two polymers with number-average molecular weight (Mn) (GPC) of 10.9 and 20.9 kDa and molecular weight dispersities (Ð) of 1.34 and 1.71, respectively. Hydrolytic degradation of the polymers was analyzed by 1H-NMR and GPC under basic and acidic conditions. The reversible covalent attachment of these polymers to lysozyme, as well as the hydrolytic and reductive cleavage of the polymer from the protein, was analyzed by gel electrophoresis and mass spectrometry. Following reductive cleavage of the polymer, an increase in activity was observed for both conjugates, with the released protein having full activity. This represents a method to prepare PEGylated proteins, where the polymer is readily cleaved from the protein and the main chain of the polymer is degradable. PMID:25937643

  6. Bubble Velocities in Slowly Sheared Bubble Rafts

    NASA Astrophysics Data System (ADS)

    Dennin, Michael

    2004-03-01

    Many complex fluids, such as foams, emulsions, colloids, and granular matter, exhibit interesting flow behavior when subjected to slow, steady rates of strain. The flow is characterized by irregular fluctuations in the stress with corresponding nonlinear rearrangements of the individual particles. We focus on the flow behavior of a model two-dimensional system: bubble rafts. Bubble rafts consist of a single layer of soap bubbles floating on the surface of a liquid subphase, usually a soap-water solution. The bubbles are sheared using a Couette geometry, i.e. concentric cylinders. We rotate the outer cylinder at a constant rate and measure the motions of individual bubbles and the stress on the inner cylinder. We will report on the velocity profiles of the bubbles averaged over long-times and averaged over individual stress events. The long-time average velocities are well described by continuum models for fluids with the one surprising feature that there exists a critical radius at which the shear-rate is discontinuous. The individual profiles are highly nonlinear and strongly correlated with the stress fluctuations. We will discuss a number of interesting questions. Can the average profiles be understood in a simple way given the individual velocities? Is there a clear "classification" for the individual profiles, or are they purely random? What sets the critical radius for a given set of flow conditions?

  7. Cholesterol's location in lipid bilayers

    DOE PAGESBeta

    Marquardt, Drew; Kučerka, Norbert; Wassall, Stephen R.; Harroun, Thad A.; Katsaras, John

    2016-04-04

    It is well known that cholesterol modifies the physical properties of lipid bilayers. For example, the much studied liquid-ordered Lo phase contains rapidly diffusing lipids with their acyl chains in the all trans configuration, similar to gel phase bilayers. Moreover, the Lo 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 is in themore » 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

  8. Immunofluorescence microscopy of SNAP23 in human skeletal muscle reveals colocalization with plasma membrane, lipid droplets, and mitochondria.

    PubMed

    Strauss, Juliette A; Shaw, Christopher S; Bradley, Helen; Wilson, Oliver J; Dorval, Thierry; Pilling, James; Wagenmakers, Anton J M

    2016-01-01

    Synaptosomal-associated protein 23 (SNAP23) is a SNARE protein expressed abundantly in human skeletal muscle. Its established role is to mediate insulin-stimulated docking and fusion of glucose transporter 4 (GLUT4) with the plasma membrane. Recent in vitro research has proposed that SNAP23 may also play a role in the fusion of growing lipid droplets (LDs) and the channeling of LD-derived fatty acids (FAs) into neighboring mitochondria for β-oxidation. This study investigates the subcellular distribution of SNAP23 in human skeletal muscle using immunofluorescence microscopy to confirm that SNAP23 localization supports the three proposed metabolic roles. Percutaneous biopsies were obtained from the m. vastus lateralis of six lean, healthy males in the rested, overnight fasted state. Cryosections were stained with antibodies targeting SNAP23, the mitochondrial marker cytochrome c oxidase and the plasma membrane marker dystrophin, whereas intramuscular LDs were stained using the neutral lipid dye oil red O. SNAP23 displayed areas of intense punctate staining in the intracellular regions of all muscle fibers and continuous intense staining in peripheral regions of the cell. Quantitation of confocal microscopy images showed colocalization of SNAP23 with the plasma membrane marker dystrophin (Pearson's correlation coefficient r = 0.50 ± 0.01). The intense punctate intracellular staining colocalized primarily with the mitochondrial marker cytochrome C oxidase (r = 0.50 ± 0.012) and to a lesser extent with LDs (r = 0.21 ± 0.01) visualized with oil red O. We conclude that the observed subcellular distribution of SNAP23 in human skeletal muscle supports the three aforementioned metabolic roles. PMID:26733245

  9. High-Affinity Binding of Remyelinating Natural Autoantibodies to Myelin-Mimicking Lipid Bilayers Revealed by Nanohole Surface Plasmon Resonance

    PubMed Central

    Wittenberg, Nathan J.; Im, Hyungsoon; Xu, Xiaohua; Wootla, Bharath; Watzlawik, Jens; Warrington, Arthur E.; Rodriguez, Moses; Oh, Sang-Hyun

    2012-01-01

    Multiple sclerosis is a progressive neurological disorder that results in the degradation of myelin sheaths that insulate axons in the central nervous system. Therefore promotion of myelin repair is a major thrust of multiple sclerosis treatment research. Two mouse monoclonal natural autoantibodies, O1 and O4, promote myelin repair in several mouse models of multiple sclerosis. Natural autoantibodies are generally polyreactive and predominantly of the IgM isotype. The prevailing paradigm is that because they are polyreactive, these antibodies bind antigens with low affinities. Despite their wide use in neuroscience and glial cell research, however, the affinities and kinetic constants of O1 and O4 antibodies have not been measured to date. In this work, we developed a membrane biosensing platform based on surface plasmon resonance in gold nanohole arrays with a series of surface modification techniques to form myelin-mimicking lipid bilayer membranes to measure both the association and dissociation rate constants for O1 and O4 antibodies binding to their myelin lipid antigens. The ratio of rate constants shows that O1 and O4 bind to galactocerebroside and sulfated galactocerebroside, respectively, with unusually small apparent dissociation constants (KD ~0.9 nM) for natural autoantibodies. This is approximately one to two orders of magnitude lower than typically observed for the highest affinity natural autoantibodies. We propose that the unusually high affinity of O1 and O4 to their targets in myelin contributes to the mechanism by which they signal oligodendrocytes and induce central nervous system repair. PMID:22762372

  10. Reconstituting ring-rafts in bud-mimicking topography of model membranes

    NASA Astrophysics Data System (ADS)

    Ryu, Yong-Sang; Lee, In-Ho; Suh, Jeng-Hun; Park, Seung Chul; Oh, Soojung; Jordan, Luke R.; Wittenberg, Nathan J.; Oh, Sang-Hyun; Jeon, Noo Li; Lee, Byoungho; Parikh, Atul N.; Lee, Sin-Doo

    2014-07-01

    During vesicular trafficking and release of enveloped viruses, the budding and fission processes dynamically remodel the donor cell membrane in a protein- or a lipid-mediated manner. In all cases, in addition to the generation or relief of the curvature stress, the buds recruit specific lipids and proteins from the donor membrane through restricted diffusion for the development of a ring-type raft domain of closed topology. Here, by reconstituting the bud topography in a model membrane, we demonstrate the preferential localization of cholesterol- and sphingomyelin-enriched microdomains in the collar band of the bud-neck interfaced with the donor membrane. The geometrical approach to the recapitulation of the dynamic membrane reorganization, resulting from the local radii of curvatures from nanometre-to-micrometre scales, offers important clues for understanding the active roles of the bud topography in the sorting and migration machinery of key signalling proteins involved in membrane budding.

  11. Reconstituting ring-rafts in bud-mimicking topography of model membranes

    PubMed Central

    Ryu, Yong-Sang; Lee, In-Ho; Suh, Jeng-Hun; Park, Seung Chul; Oh, Soojung; Jordan, Luke R.; Wittenberg, Nathan J.; Oh, Sang-Hyun; Jeon, Noo Li; Lee, Byoungho; Parikh, Atul N.; Lee, Sin-Doo

    2014-01-01

    During vesicular trafficking and release of enveloped viruses, the budding and fission processes dynamically remodel the donor cell membrane in a protein- or a lipid-mediated manner. In all cases, in addition to the generation or relief of the curvature stress, the buds recruit specific lipids and proteins from the donor membrane through restricted diffusion for the development of a ring-type raft domain of closed topology. Here, by reconstituting the bud topography in a model membrane, we demonstrate the preferential localization of cholesterol- and sphingomyelin-enriched microdomains in the collar band of the bud-neck interfaced with the donor membrane. The geometrical approach to the recapitulation of the dynamic membrane reorganization, resulting from the local radii of curvatures from nanometre-to-micrometre scales, offers important clues for understanding the active roles of the bud topography in the sorting and migration machinery of key signalling proteins involved in membrane budding. PMID:25058275

  12. Reconstituting ring-rafts in bud-mimicking topography of model membranes.

    PubMed

    Ryu, Yong-Sang; Lee, In-Ho; Suh, Jeng-Hun; Park, Seung Chul; Oh, Soojung; Jordan, Luke R; Wittenberg, Nathan J; Oh, Sang-Hyun; Jeon, Noo Li; Lee, Byoungho; Parikh, Atul N; Lee, Sin-Doo

    2014-01-01

    During vesicular trafficking and release of enveloped viruses, the budding and fission processes dynamically remodel the donor cell membrane in a protein- or a lipid-mediated manner. In all cases, in addition to the generation or relief of the curvature stress, the buds recruit specific lipids and proteins from the donor membrane through restricted diffusion for the development of a ring-type raft domain of closed topology. Here, by reconstituting the bud topography in a model membrane, we demonstrate the preferential localization of cholesterol- and sphingomyelin-enriched microdomains in the collar band of the bud-neck interfaced with the donor membrane. The geometrical approach to the recapitulation of the dynamic membrane reorganization, resulting from the local radii of curvatures from nanometre-to-micrometre scales, offers important clues for understanding the active roles of the bud topography in the sorting and migration machinery of key signalling proteins involved in membrane budding. PMID:25058275

  13. 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. PMID:21974650

  14. Rafts and the battleships of defense: the multifaceted microdomains for positive and negative signals in immune cells.

    PubMed

    Szöor, Arpád; Szöllosi, János; Vereb, György

    2010-05-01

    Recognition of the heterogeneity of the cell membrane was one of the most important scientific achievements in the last decades. Since coining the term "lipid rafts", continuous development of advanced microscopic and spectroscopic techniques has vastly expanded our view on these cell membrane microdomains that appear to have almost as many faces as researchers that look at them; they are variable in stability, size and composition that can change in a highly dynamic manner both by recruiting and expelling components as well as by coalescing and breaking up into smaller units. They have, however, one common feature: all eukaryotic cells present some variation of lipid rafts. Cells of the immune system are not exception to this, regardless of their lymphoid or myeloid origin their membranes show a domain structure and these domains serve to condense or reject particular transmembrane, GPI-linked and intracellularly membrane-anchored proteins as function requires. Here we provide a concise overview about the various weapons and shields that immune cells concentrate into their rafts, which have come into sight during the past years. The positive and negative regulatory roles of these microdomains are essential both in the functions of innate immunity and processes concatenated in the adaptive immune response. PMID:20026358

  15. 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.

  16. MALDI-mass spectrometric imaging revealing hypoxia-driven lipids and proteins in a breast tumor model

    SciTech Connect

    Lu, Jiang; Chughtai, Kamila; Purvine, Samuel O.; Bhujwalla, Zaver M.; Raman, Venu; Pasa-Tolic, Ljiljana; Heeren, Ronald M.; Glunde, Kristine

    2015-06-16

    Hypoxic areas are a common feature of rapidly growing malignant tumors and their metastases, and are typically spatially heterogeneous. Hypoxia has a strong impact on tumor cell biology and contributes to tumor progression in multiple ways. To date, only a few molecular key players in tumor hypoxia, such as for example hypoxia-inducible factor-1 (HIF-1), have been discovered. The distribution of biomolecules is frequently heterogeneous in the tumor volume, and may be driven by hypoxia and HIF-1α. Understanding the spatially heterogeneous hypoxic response of tumors is critical. Mass spectrometric imaging (MSI) provides a unique way of imaging biomolecular distributions in tissue sections with high spectral and spatial resolution. In this paper, breast tumor xenografts grown from MDA-MB-231-HRE-tdTomato cells, with a red fluorescent tdTomato protein construct under the control of a hypoxia response element (HRE)-containing promoter driven by HIF-1α, were used to detect the spatial distribution of hypoxic regions. We elucidated the 3D spatial relationship between hypoxic regions and the localization of small molecules, metabolites, lipids, and proteins by using principal component analysis – linear discriminant analysis (PCA-LDA) on 3D rendered MSI volume data from MDA-MB-231-HRE-tdTomato breast tumor xenografts. In this study we identified hypoxia-regulated proteins active in several distinct pathways such as glucose metabolism, regulation of actin cytoskeleton, protein folding, translation/ribosome, splicesome, the PI3K-Akt signaling pathway, hemoglobin chaperone, protein processing in endoplasmic reticulum, detoxification of reactive oxygen species, aurora B signaling/apoptotic execution phase, the RAS signaling pathway, the FAS signaling pathway/caspase cascade in apoptosis and telomere stress induced senescence. In parallel we also identified co-localization of hypoxic regions and various lipid species such as PC(16:0/18:1), PC(16:0/18:2), PC(18:0/18:1), PC

  17. Transcriptome Analysis of the Oil-Rich Tea Plant, Camellia oleifera, Reveals Candidate Genes Related to Lipid Metabolism

    PubMed Central

    Huang, Hui; Zhang, Li-Ping; Zhang, Hai-Bin; Gao, Li-Zhi

    2014-01-01

    Background Rapidly driven by the need for developing sustainable sources of nutritionally important fatty acids and the rising concerns about environmental impacts after using fossil oil, oil-plants have received increasing awareness nowadays. As an important oil-rich plant in China, Camellia oleifera has played a vital role in providing nutritional applications, biofuel productions and chemical feedstocks. However, the lack of C. oleifera genome sequences and little genetic information have largely hampered the urgent needs for efficient utilization of the abundant germplasms towards modern breeding efforts of this woody oil-plant. Results Here, using the 454 GS-FLX sequencing platform, we generated approximately 600,000 RNA-Seq reads from four tissues of C. oleifera. These reads were trimmed and assembled into 104,842 non-redundant putative transcripts with a total length of ∼38.9 Mb, representing more than 218-fold of all the C. oleifera sequences currently deposited in the GenBank (as of March 2014). Based on the BLAST similarity searches, nearly 42.6% transcripts could be annotated with known genes, conserved domains, or Gene Ontology (GO) terms. Comparisons with the cultivated tea tree, C. sinensis, identified 3,022 pairs of orthologs, of which 211 exhibited the evidence under positive selection. Pathway analysis detected the majority of genes potentially related to lipid metabolism. Evolutionary analysis of omega-6 fatty acid desaturase (FAD2) genes among 20 oil-plants unexpectedly suggests that a parallel evolution may occur between C. oleifera and Olea oleifera. Additionally, more than 2,300 simple sequence repeats (SSRs) and 20,200 single-nucleotide polymorphisms (SNPs) were detected in the C. oleifera transcriptome. Conclusions The generated transcriptome represents a considerable increase in the number of sequences deposited in the public databases, providing an unprecedented opportunity to discover all related-genes associated with lipid metabolic

  18. Reduction in Tension and Stiffening of Lipid Membranes in an Electric Field Revealed by X-Ray Scattering

    NASA Astrophysics Data System (ADS)

    Hemmerle, Arnaud; Fragneto, Giovanna; Daillant, Jean; Charitat, Thierry

    2016-06-01

    The effect of ac electric fields on the elasticity of supported lipid bilayers is investigated at the microscopic level using grazing incidence synchrotron x-ray scattering. A strong decrease in the membrane tension up to 1 mN /m and a dramatic increase of its effective rigidity up to 300 kBT are observed for local electric potentials seen by the membrane ≲1 V . The experimental results are analyzed using detailed electrokinetic modeling and nonlinear Poisson-Boltzmann theory. Based on a modeling of the electromagnetic stress, which provides an accurate description of the bilayer separation versus pressure curves, we show that the decrease in tension results from the amplification of charge fluctuations on the membrane surface whereas the increase in bending rigidity results from the direct interaction between charges in the electric double layer. These effects eventually lead to a destabilization of the bilayer and vesicle formation. Similar effects are expected at the tens of nanometers length scale in cell membranes with lower tension, and could explain a number of electrically driven processes.

  19. Quantitative proteomics analysis of the liver reveals immune regulation and lipid metabolism dysregulation in a mouse model of depression.

    PubMed

    Wu, You; Tang, Jianyong; Zhou, Chanjuan; Zhao, Libo; Chen, Jin; Zeng, Li; Rao, Chenglong; Shi, Haiyang; Liao, Li; Liang, Zihong; Yang, Yongtao; Zhou, Jian; Xie, Peng

    2016-09-15

    Major depressive disorder (MDD) is a highly prevalent and debilitating mental illness with substantial impairments in quality of life and functioning. However, the pathophysiology of major depression remains poorly understood. Combining the brain and body should provide a comprehensive understanding of the etiology of MDD. As the largest internal organ of the human body, the liver has an important function, yet no proteomic study has assessed liver protein expression in a preclinical model of depression. Using the chronic unpredictable mild stress (CUMS) mouse model of depression, differential protein expression between CUMS and control (CON) mice was examined in the liver proteome using isobaric tag for relative and absolute quantitation (iTRAQ) coupled with tandem mass spectrometry. More than 4000 proteins were identified and 66 most significantly differentiated proteins were used for further bioinformatic analysis. According to the ingenuity pathway analysis (IPA), we found that proteins related to the inflammation response, immune regulation, lipid metabolism and NFκB signaling network were altered by CUMS. Moreover, four proteins closely associated with these processes, hemopexin, haptoglobin, cytochrome P450 2A4 (CYP2A4) and bile salt sulfotransferase 1 (SULT2A1), were validated by western blotting. In conclusion, we report, for the first time, the liver protein expression profile in the CUMS mouse model of depression. Our findings provide novel insight (liver-brain axis) into the multifaceted mechanisms of major depressive disorder. PMID:27247144

  20. Low temporal variation in the intact polar lipid composition of North Sea coastal marine water reveals limited chemotaxonomic value

    NASA Astrophysics Data System (ADS)

    Brandsma, J.; Hopmans, E. C.; Philippart, C. J. M.; Veldhuis, M. J. W.; Schouten, S.; Sinninghe Damsté, J. S.

    2012-03-01

    Temporal variations in the abundance and composition of intact polar lipids (IPLs) in North Sea coastal marine water were assessed over a one-year seasonal cycle, and compared with environmental parameters and the microbial community composition. Sulfoquinovosyldiacylglycerol (SQDG) was the most abundant IPL class, followed by phosphatidylcholine (PC), phosphatidylglycerol (PG) and diacylglyceryl-(N,N,N)-trimethylhomoserine (DGTS) in roughly equal concentrations, and smaller amounts of phosphatidylethanolamine (PE). Although the total concentrations of these IPL classes varied substantially throughout the year, the composition of the IPL pool remained remarkably constant. Statistical analysis yielded negative correlations between IPL concentrations and dissolved inorganic nutrient concentrations, but no changes in the overall planktonic IPL composition due to nutrient limitation were observed. Significant correlations between SQDG, PC, PG and DGTS concentrations and chlorophyll a concentrations and algal abundances indicated that eukaryotic primary producers, in particular Phaeocystis globosa, were the predominant source of IPLs at this site. However, while IPL concentrations in the water were closely tied to total algal abundances, the rapid succession of different algal groups blooming throughout the year resulted in only minor shifts in the IPL composition. Principal component analysis showed that the IPLs were derived from multiple sources, and that no IPL species could be exclusively assigned to a particular algal taxa or (cyano)bacteria. Thus, the most commonly occurring IPLs appear to have limited chemotaxonomic potential, highlighting the need to use targeted assays of more specific biomarker IPLs.

  1. Reduction in Tension and Stiffening of Lipid Membranes in an Electric Field Revealed by X-Ray Scattering.

    PubMed

    Hemmerle, Arnaud; Fragneto, Giovanna; Daillant, Jean; Charitat, Thierry

    2016-06-01

    The effect of ac electric fields on the elasticity of supported lipid bilayers is investigated at the microscopic level using grazing incidence synchrotron x-ray scattering. A strong decrease in the membrane tension up to 1  mN/m and a dramatic increase of its effective rigidity up to 300  k_{B}T are observed for local electric potentials seen by the membrane ≲1  V. The experimental results are analyzed using detailed electrokinetic modeling and nonlinear Poisson-Boltzmann theory. Based on a modeling of the electromagnetic stress, which provides an accurate description of the bilayer separation versus pressure curves, we show that the decrease in tension results from the amplification of charge fluctuations on the membrane surface whereas the increase in bending rigidity results from the direct interaction between charges in the electric double layer. These effects eventually lead to a destabilization of the bilayer and vesicle formation. Similar effects are expected at the tens of nanometers length scale in cell membranes with lower tension, and could explain a number of electrically driven processes. PMID:27314739

  2. Stable carbon isotopic compositions of intact polar lipids reveal complex carbon flow patterns among hydrocarbon degrading microbial communities at the Chapopote asphalt volcano

    NASA Astrophysics Data System (ADS)

    Schubotz, Florence; Lipp, Julius S.; Elvert, Marcus; Hinrichs, Kai-Uwe

    2011-08-01

    Seepage of asphalt forms the basis of a cold seep system at 3000 m water depth at the Chapopote Knoll in the southern Gulf of Mexico. Anaerobic microbial communities are stimulated in the oil-impregnated sediments as evidenced by the presence of intact polar membrane lipids (IPLs) derived from archaea and Bacteria at depths up to 7 m below the seafloor. Detailed investigation of stable carbon isotope composition (δ 13C) of alkyl and acyl moieties derived from a range of IPL precursors with distinct polar head groups resolved the complexity of carbon metabolisms and utilization of diverse carbon sources by uncultured microbial communities. In surface sediments most of the polar lipid-derived fatty acids with phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) head groups could be tentatively assigned to autotrophic sulfate-reducing bacteria, with a relatively small proportion involved in the anaerobic oxidation of methane. Derivatives of phosphatidyl-( N)-methylethanolamine (PME) were abundant and could be predominantly assigned to heterotrophic oil-degrading bacteria. Archaeal IPLs with phosphate-based hydroxyarchaeols and diglycosidic glyceroldibiphytanylglyceroltetraethers (GDGTs) were assigned to methanotrophic archaea of the ANME-2 and ANME-1 cluster, respectively, whereas δ 13C values of phosphate-based archaeols and mixed phosphate-based and diglycosidic GDGTs point to methanogenic archaea. At a 7 m deep sulfate-methane transition zone that is linked to the upward movement of gas-laden petroleum, a distinct increase in abundance of archaeal IPLs such as phosphate-based hydroxyarchaeols and diglycosidic archaeol and GDGTs is observed; their δ 13C values are consistent with their origin from both methanotrophic and methanogenic archaea. This study reveals previously hidden, highly complex patterns in the carbon-flow of versatile microbial communities involved in the degradation of heavy oil including hydrocarbon gases

  3. RNAi screens reveal novel metabolic regulators: RIP140, MAP4k4 and the lipid droplet associated fat specific protein (FSP) 27.

    PubMed

    Puri, V; Virbasius, J V; Guilherme, A; Czech, M P

    2008-01-01

    Adipose tissue modulates whole body metabolism and insulin sensitivity by controlling circulating lipid levels and producing molecules that can regulate fatty acid metabolism in such tissues as muscle and liver. We have developed RNA interference (RNAi) screens to identify genes in cultured adipocytes that regulate insulin signalling and key metabolic pathways. These short interfering RNA (siRNA)-based screens identified the transcriptional corepressor receptor interacting protein 140 (RIP140) (J Clin Invest 116: 125, 2006) and the mitogen-activated protein kinase (MAP4k4) (Proc Natl Acad Sci USA 103: 2087, 2006) as negative regulators of insulin-responsive hexose uptake and oxidative metabolism. Gene expression profiling revealed that RIP140 depletion upregulates the expression of clusters of genes in the pathways of glucose uptake, glycolysis, tricarboxylic acid cycle, fatty acid oxidation, mitochondrial biogenesis and oxidative phosphorylation. RIP140-null mice resist weight gain on a high-fat diet and display enhanced glucose tolerance. MAP4k4 depletion in adipocytes increases many of the RIP140-sensitive genes, increases adipogenesis and mediates some actions of tumour necrosis factor-alpha (TNF-alpha). Remarkably, another hit in our RNAi screens was fat specific protein 27 (FSP27), a highly expressed isoform of Cidea. We discovered that FSP27 unexpectedly associates specifically with lipid droplets and regulates fat storage. We conclude that RIP140, MAP4k4 and the novel lipid droplet protein FSP27 are powerful regulators of adipose tissue metabolism and are potential therapeutic targets for controlling metabolic disease. The discovery of these novel proteins validates the power of RNAi screening for discovery of new therapeutic approaches to type 2 diabetes and obesity. PMID:18171433

  4. Adaptive Lipid Packing and Bioactivity in Membrane Domains

    PubMed Central

    Sezgin, Erdinc; Gutmann, Theresia; Buhl, Tomasz; Dirkx, Ron; Grzybek, Michal; Coskun, Ünal; Solimena, Michele; Simons, Kai; Levental, Ilya; Schwille, Petra

    2015-01-01

    Lateral compositional and physicochemical heterogeneity is a ubiquitous feature of cellular membranes on various length scales, from molecular assemblies to micrometric domains. Segregated lipid domains of increased local order, referred to as rafts, are believed to be prominent features in eukaryotic plasma membranes; however, their exact nature (i.e. size, lifetime, composition, homogeneity) in live cells remains difficult to define. Here we present evidence that both synthetic and natural plasma membranes assume a wide range of lipid packing states with varying levels of molecular order. These states may be adapted and specifically tuned by cells during active cellular processes, as we show for stimulated insulin secretion. Most importantly, these states regulate both the partitioning of molecules between coexisting domains and the bioactivity of their constituent molecules, which we demonstrate for the ligand binding activity of the glycosphingolipid receptor GM1. These results confirm the complexity and flexibility of lipid-mediated membrane organization and reveal mechanisms by which this flexibility could be functionalized by cells. PMID:25905447

  5. Lipid domains in supported SM-Chol membranes measured by GISANS

    NASA Astrophysics Data System (ADS)

    Zhernenkov, Mikhail; Dubey, Manish; Toperverg, Boris; Majewski, Jaroslaw; Fitzsimmons, Michael

    2011-03-01

    Cell membranes are known to contain regions (called lipid domains, or rafts) described as sphingolipid-cholesterol assemblies which also may contain a subset of membrane proteins. Currently, the main point of discussion is the methodology to study lipid domains and their sizes. We report on Grazing Incidence Small Angle Neutron Scattering (GISANS) measurements of lipid domains in supported sphingomyelin(SM)-cholesterol(Chol) bilayers in a fully aqueous environment. The model bilayers SM:Chol(2:1), SM:Chol(1:2), and a pure SM were deposited using Langmuir-Blodgett/Langmuir-Schaefer technique at a surface pressure of 10 mN/m and measured at 25rC. First measurements revealed short range inhomogeneities of the order of 100 AA in both binary systems. The control measurement of a pure SM bilayer exhibited nearly no GISANS indicating an absence of lipid domains in the SM bilayer. This observation is consistent with the notion that a single component system studied below the liquid-gel transition temperature will not produce lipid domains. Work was supported by DOE-BES.

  6. Metabolomics revealed diurnal heat stress and zinc supplementation-induced changes in amino acid, lipid, and microbial metabolism.

    PubMed

    Wang, Lei; Urriola, Pedro E; Luo, Zhao-Hui; Rambo, Zachary J; Wilson, Mark E; Torrison, Jerry L; Shurson, Gerald C; Chen, Chi

    2016-01-01

    Heat stress (HS) dramatically disrupts the events in energy and nutrient metabolism, many of which requires zinc (Zn) as a cofactor. In this study, metabolic effects of HS and Zn supplementation were evaluated by examining growth performance, blood chemistry, and metabolomes of crossbred gilts fed with ZnNeg (no Zn supplementation), ZnIO (120 ppm ZnSO4), or ZnAA (60 ppm ZnSO4 + 60 ppm zinc amino acid complex) diets under diurnal HS or thermal-neutral (TN) condition. The results showed that growth performance was reduced by HS but not by Zn supplementation. Among measured serum biochemicals, HS was found to increase creatinine but decrease blood urea nitrogen (BUN) level. Metabolomic analysis indicated that HS greatly affected diverse metabolites associated with amino acid, lipid, and microbial metabolism, including urea cycle metabolites, essential amino acids, phospholipids, medium-chain dicarboxylic acids, fatty acid amides, and secondary bile acids. More importantly, many changes in these metabolite markers were correlated with both acute and adaptive responses to HS. Relative to HS-induced metabolic effects, Zn supplementation-associated effects were much more limited. A prominent observation was that ZnIO diet, potentially through its influences on microbial metabolism, yielded different responses to HS compared with two other diets, which included higher levels of short-chain fatty acids (SCFAs) in cecal fluid and higher levels of lysine in the liver and feces. Overall, comprehensive metabolomic analysis identified novel metabolite markers associated with HS and Zn supplementation, which could guide further investigation on the mechanisms of these metabolic effects. PMID:26755737

  7. Structure-Based Design of an Organoruthenium Phosphatidyl-inositol-3-Kinase Inhibitor Reveals a Switch Governing Lipid Kinase Potency and Selectivity

    SciTech Connect

    Xie,P.; Williams, D.; Atilla-Gokcumen, G.; Milk, L.; Xiao, M.; Smalley, K.; Herlyn, M.; Meggers, E.; Marmorstein, R.

    2008-01-01

    Mutations that constitutively activate the phosphatidyl-inositol-3-kinase (PI3K) signaling pathway, including alterations in PI3K, PTEN, and AKT, are found in a variety of human cancers, implicating the PI3K lipid kinase as an attractive target for the development of therapeutic agents to treat cancer and other related diseases. In this study, we report on the combination of a novel organometallic kinase inhibitor scaffold with structure-based design to develop a PI3K inhibitor, called E5E2, with an IC50 potency in the mid-low-nanomolar range and selectivity against a panel of protein kinases. We also show that E5E2 inhibits phospho-AKT in human melanoma cells and leads to growth inhibition. Consistent with a role for the PI3K pathway in tumor cell invasion, E5E2 treatment also inhibits the migration of melanoma cells in a 3D spheroid assay. The