Targeting of ion channels to membrane microdomains: localization of KV channels to lipid rafts

2004 ◽  
Vol 25 (1) ◽  
pp. 16-21 ◽  
Author(s):  
Jeffrey R Martens ◽  
Kristen O'Connell ◽  
Michael Tamkun
Keyword(s):  
Ion Channels ◽  
Lipid Rafts ◽  
Membrane Microdomains ◽  
Kv Channels

scholarly journals Distribution of a Glycosylphosphatidylinositol-anchored Protein at the Apical Surface of MDCK Cells Examined at a Resolution of <100 Å Using Imaging Fluorescence Resonance Energy Transfer

1998 ◽  
Vol 142 (1) ◽  
pp. 69-84 ◽  
Author(s):  
A.K. Kenworthy ◽  
M. Edidin
Keyword(s):  
Energy Transfer ◽  
Lipid Rafts ◽  
Fluorescence Resonance Energy Transfer ◽  
Mdck Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Microdomains ◽  
Apical Surface ◽  
Donor And Acceptor ◽  
Fluorescence Resonance

Membrane microdomains (“lipid rafts”) enriched in glycosylphosphatidylinositol (GPI)-anchored proteins, glycosphingolipids, and cholesterol have been implicated in events ranging from membrane trafficking to signal transduction. Although there is biochemical evidence for such membrane microdomains, they have not been visualized by light or electron microscopy. To probe for microdomains enriched in GPI- anchored proteins in intact cell membranes, we used a novel form of digital microscopy, imaging fluorescence resonance energy transfer (FRET), which extends the resolution of fluorescence microscopy to the molecular level (&lt;100 Å). We detected significant energy transfer between donor- and acceptor-labeled antibodies against the GPI-anchored protein 5′ nucleotidase (5′ NT) at the apical membrane of MDCK cells. The efficiency of energy transfer correlated strongly with the surface density of the acceptor-labeled antibody. The FRET data conformed to theoretical predictions for two-dimensional FRET between randomly distributed molecules and were inconsistent with a model in which 5′ NT is constitutively clustered. Though we cannot completely exclude the possibility that some 5′ NT is in clusters, the data imply that most 5′ NT molecules are randomly distributed across the apical surface of MDCK cells. These findings constrain current models for lipid rafts and the membrane organization of GPI-anchored proteins.

scholarly journals Pseudotypes of Vesicular Stomatitis Virus with CD4 Formed by Clustering of Membrane Microdomains during Budding

2005 ◽  
Vol 79 (11) ◽  
pp. 7077-7086 ◽  
Author(s):  
Erica L. Brown ◽  
Douglas S. Lyles
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Lipid Rafts ◽  
Immunoelectron Microscopy ◽  
Vesicular Stomatitis Virus ◽  
Plasma Membranes ◽  
Membrane Microdomains ◽  
Virus Budding ◽  
Vesicular Stomatitis ◽  
Membrane Components

ABSTRACT Many plasma membrane components are organized into detergent-resistant membrane microdomains referred to as lipid rafts. However, there is much less information about the organization of membrane components into microdomains outside of lipid rafts. Furthermore, there are few approaches to determine whether different membrane components are colocalized in microdomains as small as lipid rafts. We have previously described a new method of determining the extent of organization of proteins into membrane microdomains by analyzing the distribution of pairwise distances between immunogold particles in immunoelectron micrographs. We used this method to analyze the microdomains involved in the incorporation of the T-cell antigen CD4 into the envelope of vesicular stomatitis virus (VSV). In cells infected with a recombinant virus that expresses CD4 from the viral genome, both CD4 and the VSV envelope glycoprotein (G protein) were found in detergent-soluble (nonraft) membrane fractions. However, analysis of the distribution of CD4 and G protein in plasma membranes by immunoelectron microscopy showed that both were organized into membrane microdomains of similar sizes, approximately 100 to 150 nm. In regions of plasma membrane outside of virus budding sites, CD4 and G protein were present in separate membrane microdomains, as shown by double-label immunoelectron microscopy data. However, virus budding occurred from membrane microdomains that contained both G protein and CD4, and extended to approximately 300 nm, indicating that VSV pseudotype formation with CD4 occurs by clustering of G protein- and CD4-containing microdomains.

Membrane microdomains and proteomics: Lessons from tetraspanin microdomains and comparison with lipid rafts

PROTEOMICS ◽  
2006 ◽  
Vol 6 (24) ◽  
pp. 6447-6454 ◽  
Author(s):  
François Le Naour ◽  
Magali André ◽  
Claude Boucheix ◽  
Eric Rubinstein
Keyword(s):  
Lipid Rafts ◽  
Membrane Microdomains

Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation

2002 ◽  
Vol 115 (12) ◽  
pp. 2603-2611 ◽  
Author(s):  
Martha Triantafilou ◽  
Kensuke Miyake ◽  
Douglas T. Golenbock ◽  
Kathy Triantafilou
Keyword(s):  
Lipid Rafts ◽  
Lipid Raft ◽  
Cell Activation ◽  
Imaging Techniques ◽  
Recognition System ◽  
Innate Immune ◽  
Membrane Microdomains ◽  
Immune Recognition ◽  
Cellular Activation ◽  
Signalling Molecules

The plasma membrane of cells is composed of lateral heterogeneities,patches and microdomains. These membrane microdomains or lipid rafts are enriched in glycosphingolipids and cholesterol and have been implicated in cellular processes such as membrane sorting and signal transduction. In this study we investigated the importance of lipid raft formation in the innate immune recognition of bacteria using biochemical and fluorescence imaging techniques. We found that receptor molecules that are implicated in lipopolysaccharide (LPS)-cellular activation, such as CD14, heat shock protein(hsp) 70, 90, Chemokine receptor 4 (CXCR4), growth differentiation factor 5(GDF5) and Toll-like receptor 4 (TLR4), are present in microdomains following LPS stimulation. Lipid raft integrity is essential for LPS-cellular activation, since raft-disrupting drugs, such as nystatin or MCD, inhibit LPS-induced TNF-α secretion. Our results suggest that the entire bacterial recognition system is based around the ligation of CD14 by bacterial components and the recruitment of multiple signalling molecules, such as hsp70, hsp90, CXCR4, GDF5 and TLR4, at the site of CD14-LPS ligation, within the lipid rafts.

scholarly journals Palmitoylation of the Autographa californica Multicapsid Nucleopolyhedrovirus Envelope Glycoprotein GP64: Mapping, Functional Studies, and Lipid Rafts

2003 ◽  
Vol 77 (11) ◽  
pp. 6265-6273 ◽  
Author(s):  
Sandy Xiaoxin Zhang ◽  
Yu Han ◽  
Gary W. Blissard
Keyword(s):  
Cell Surface ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Envelope Glycoprotein ◽  
Cysteine Residue ◽  
Autographa Californica ◽  
Membrane Microdomains ◽  
Sf9 Cells ◽  
Wild Type ◽  
Lipid Raft Microdomains

ABSTRACT Budded virions (BV) of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) contain a major envelope glycoprotein known as GP64, which was previously shown to be palmitoylated. In the present study, we used truncation and amino acid substitution mutations to map the palmitoylation site to cysteine residue 503. Palmitoylation of GP64 was not detected when Cys503 was replaced with alanine or serine. Palmitoylation-minus forms of GP64 were used to replace wild-type GP64 in AcMNPV, and these viruses were used to examine potential functions of GP64 palmitoylation in the context of the infection cycle. Analysis by immunoprecipitation and cell surface studies revealed that palmitoylation of GP64 did not affect GP64 synthesis or its transport to the cell surface in Sf9 cells. GP64 proteins lacking palmitoylation also mediated low-pH-triggered membrane fusion in a manner indistinguishable from that of wild-type GP64. Cells infected with viruses expressing palmitoylation-minus forms of GP64 produced infectious virions at levels similar to those from cells infected with wild-type AcMNPV. In combination, these data suggest that virus entry and exit in Sf9 cells were not significantly affected by GP64 palmitoylation. To determine whether GP64 palmitoylation affected the association of GP64 with membrane microdomains, the potential association of GP64 with lipid raft microdomains was examined. These experiments showed that: (i) AcMNPV-infected Sf9 cell membranes contain lipid raft microdomains, (ii) GP64 association with lipid rafts was not detected in infected Sf9 cells, and (iii) GP64 palmitoylation did not affect the apparent exclusion of GP64 from lipid raft microdomains.

Lipid raft adhesion receptors and Syk regulate selectin-dependent rolling under flow conditions

Blood ◽  
2006 ◽  
Vol 108 (10) ◽  
pp. 3352-3359 ◽  
Author(s):  
Claire Abbal ◽  
Martine Lambelet ◽  
Debora Bertaggia ◽  
Carole Gerbex ◽  
Manuel Martinez ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Chelating Agents ◽  
Regulatory Mechanisms ◽  
Membrane Microdomains ◽  
Leukocyte Rolling ◽  
Flow Conditions ◽  
Cell Rolling ◽  
Pharmacologic Inhibitors

Abstract Selectins and their ligand P-selectin glycoprotein ligand-1 (PSGL-1) mediate leukocyte rolling along inflamed vessels. Cell rolling is modulated by selectin interactions with their ligands and by topographic requirements including L-selectin and PSGL-1 clustering on tips of leukocyte microvilli. Lipid rafts are cell membrane microdomains reported to function as signaling platforms. Here, we show that disruption of leukocyte lipid rafts with cholesterol chelating agents depleted raft-associated PSGL-1 and L-selectin and strongly reduced L-, P-, and E-selectin–dependent rolling. Cholesterol repletion reversed inhibition of cell rolling. Importantly, leukocyte rolling on P-selectin induced the recruitment of spleen tyrosine kinase (Syk), a tyrosine kinase associated to lipid raft PSGL-1. Furthermore, inhibition of Syk activity or expression, with pharmacologic inhibitors or by RNA interference, strongly reduced leukocyte rolling on P-selectin, but not on E-selectin or PSGL-1. These observations identify novel regulatory mechanisms of leukocyte rolling on selectins with a strong dependency on lipid raft integrity and Syk activity.

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

2013 ◽  
Vol 16 (6) ◽  
pp. 1361-1371 ◽  
Author(s):  
Caroline Nothdurfter ◽  
Sascha Tanasic ◽  
Barbara Di Benedetto ◽  
Manfred Uhr ◽  
Eva-Maria Wagner ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Lipid Rafts ◽  
Gabaa Receptor ◽  
Lipid Raft ◽  
Tricyclic Antidepressant ◽  
Membrane Microdomains ◽  
Cholesterol Depletion ◽  
Receptor Modulation ◽  
Gabaa Receptor Subunits ◽  
Triton X

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

Abstract 488: Sinoatrial Nodal Pacemaker Cells (SANC) Exhibit High Basal CA Activated Adenylyl Cyclase (AC) Activity and Express Multiple Distinctly Localized AC Types

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
David R Graham ◽  
Antoine Younes ◽  
Alexey Lyashkov ◽  
Anna Sheydina ◽  
Maria Volkova ◽  
...  
Keyword(s):  
Lipid Rafts ◽  
Sucrose Gradient ◽  
Soluble Fraction ◽  
Membrane Microdomains ◽  
Membrane Domains ◽  
Lipid Domains ◽  
Rt Pcr ◽  
Pacemaker Cells ◽  
Pacemaker Function ◽  
Sucrose Gradient Ultracentrifugation

In SANC constituitive AC generates high basal cAMP, inducing PKA-dependent phosphorylation that regulates Ca2+ cycling, that is essential for normal pacemaker function. Our goals were to identify, in rabbit SANC, the types of AC expressed, and their Ca2+ sensitivity and location. Radioimmunoassay (with total phosphodiasterase inhibition) showed a high Ca2+ activated basal AC activity. AC activity increased 5-fold from Ca2+ free (EGTA) to 1 uM free Ca2+. RT PCR (using specifically designed rabbit primers) showed that AC types II and V, and Ca2+ activated types, I and VIII, are expressed in SANC. The organization of these distinct AC types within calveolar or non-calveolar membrane microdomains was determined in pooled SANC isolated from 5 hearts, using triton x100, and sucrose gradient ultracentrifugation. Lipid domains segregated into caveolin containing and non-caveolin containing membrane microdomains, where AC activity was concentrated (fig , AC activity). Immunoblots demonstrated localization of different AC types between these two membrane domains, with AC I, II, V/VI localizing to caveolin containing lipid rafts, and AC VIII present in both caveolin and GM1 lipid domains, and also in the soluble fraction (fig ). In summary, multiple ACs, both Ca2+ activated and non-CA2+ activated types, are expressed in SANC, and these reside in distinct calveolar and non-calveolar lipid domains. We conclude that constituitive basal AC activity is, generated, in part, at least, by a Ca2+ activated AC. type.

scholarly journals Markers for Detergent-resistant Lipid Rafts Occupy Distinct and Dynamic Domains in Native Membranes

2004 ◽  
Vol 15 (6) ◽  
pp. 2580-2592 ◽  
Author(s):  
Bridget S. Wilson ◽  
Stanly L. Steinberg ◽  
Karin Liederman ◽  
Janet R. Pfeiffer ◽  
Zurab Surviladze ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Lipid Rafts ◽  
Coated Vesicle ◽  
Membrane Microdomains ◽  
Cross Linking ◽  
Vesicle Budding ◽  
Unsaturated Lipids ◽  
Dense Membrane ◽  
Ige Receptors ◽  
Gradient Fractionation

Lipid rafts isolated by detergent extraction and sucrose gradient fractionation from mast cells are enriched for the glycosylphosphatidylinositol-linked protein Thy-1, the ganglioside GM1, palmitoylated LAT, and cross-linked IgE receptors, FcϵRI. This study addresses the relationship of fractionation data to the organization of raft markers in native membranes. Immunogold labeling and electron microscopy shows there is little or no colocalization of the raft markers Thy-1, GM1, and LAT with each other or with FcϵRI on native membrane sheets prepared from unstimulated cells. External cross-linking of Thy-1 promotes coclustering of Thy-1 with LAT, but not with GM1. Thy-1 and LAT clusters occur on membrane regions without distinctive features. In contrast, external cross-linking of FcϵRI and GM1 causes their redistribution to electron-dense membrane patches independently of each other and of Thy-1. The distinctive patches that accumulate cross-linked FcϵRI and GM1 also accumulate osmium, a stain for unsaturated lipids, and are sites for coated vesicle budding. Electron microscopy reveals a more complex and dynamic topographical organization of membrane microdomains than is predicted by biochemical analysis of detergent-resistant membranes.

scholarly journals The Facultative Role of Lipid Rafts and Membrane Microdomains in Controlling the Assembly of the Store-Operated Channel Complex

2011 ◽  
Vol 100 (3) ◽  
pp. 36a
Author(s):  
Luis D. Vaca ◽  
Alexander N. Asanov ◽  
Angélica Zepeda
Keyword(s):  
Lipid Rafts ◽  
Membrane Microdomains
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