Lateral Distribution of the Transmembrane Domain of Influenza Virus Hemagglutinin Revealed by Time-resolved Fluorescence Imaging

Scolari, Silvia; Engel, Stephanie; Krebs, Nils; Plazzo, Anna Pia; Almeida, Rodrigo F.M. de; Prieto, Manuel; Veit, Michael; Herrmann, Andreas

doi:10.1074/jbc.m900437200

Cited by 76 publications

(110 citation statements)

References 41 publications

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“…Thus, the plasma membrane fraction from WT cells was isolated, and the fluorescence properties of t-PnA labeling these membranes were characterized. It was previously shown that in mammalian (Chinese hamster ovary) cells the plasma membrane isolated by ultracentrifugation retains to a very significant extent the domain organization of the plasma membrane in intact cells (32). As can be seen in Fig.…”

Section: Plasma Membrane Of S Cerevisiae Contains Highly Orderedsupporting

confidence: 50%

Gel Domains in the Plasma Membrane of Saccharomyces cerevisiae

Aresta‐Branco

Cordeiro

Marinho

et al. 2011

Journal of Biological Chemistry

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The plasma membrane of Saccharomyces cerevisiae was studied using the probes trans-parinaric acid and diphenylhexatriene. Diphenylhexatriene anisotropy is a good reporter of global membrane order. The fluorescence lifetimes of transparinaric acid are particularly sensitive to the presence and nature of ordered domains, but thus far they have not been measured in yeast cells. A long lifetime typical of the gel phase (>30 ns) was found in wild-type (WT) cells from two different genetic backgrounds, at 24 and 30°C, providing the first direct evidence for the presence of gel domains in living cells. To understand their nature and location, the study of WT cells was extended to spheroplasts, the isolated plasma membrane, and liposomes from total lipid and plasma membrane lipid extracts (with or without ergosterol extraction by cyclodextrin). It is concluded that the plasma membrane is mostly constituted by ordered domains and that the gel domains found in living cells are predominantly at the plasma membrane and are formed by lipids. To understand their composition, strains with mutations in sphingolipid and ergosterol metabolism and in the glycosylphosphatidylinositol anchor remodeling pathway were also studied. The results strongly indicate that the gel domains are not ergosterol-enriched lipid rafts; they are mainly composed of sphingolipids, possibly inositol phosphorylceramide, and contain glycosylphosphatidylinositol-anchored proteins, suggesting an important role in membrane traffic and signaling, and interactions with the cell wall. The abundance of the sphingolipid-enriched gel domains was inversely related to the cellular membrane system global order, suggesting their involvement in the regulation of membrane properties.

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Section: Plasma Membrane Of S Cerevisiae Contains Highly Orderedsupporting

confidence: 50%

Gel Domains in the Plasma Membrane of Saccharomyces cerevisiae

Aresta‐Branco

Cordeiro

Marinho

et al. 2011

Journal of Biological Chemistry

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“…We generated constructs expressing mNonFP fused to the C-terminal 72 amino acid residues of influenza virus hemagglutinin (HA) including the transmembrane and cytoplasmic domains (NonFP-HA-TMD) based on previously described constructs, a kind gift from A. Herrmann (122). A plasmid encoding CD81 with mVenus (YFP) inserted into its large extracellular loop (CD81-EC2-iYFP) was created using CD81 coding sequence from CD81-GFP (a kind gift from F. Sánchez-Madrid) (45).…”

Section: Plasmidsmentioning

confidence: 99%

“…To determine whether FRET is highly dependent on YFP concentration, indicating no specific clustering of microdomain markers, or whether FRET is less dependent on YFP concentration, indicating clustering of markers, we used a previously described method of analysis (33,72,122,142). YFP fluorescence intensity was measured for each cell using ImageJ by taking the average pixel intensity within the ROIs determined as described above.…”

Section: Plasmidsmentioning

confidence: 99%

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Gag Induces the Coalescence of Clustered Lipid Rafts and Tetraspanin-Enriched Microdomains at HIV-1 Assembly Sites on the Plasma Membrane

Hogue

Grover

Soheilian

et al. 2011

J Virol

108

116

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The HIV-1 structural protein Gag associates with two types of plasma membrane microdomains, lipid rafts and tetraspanin-enriched microdomains (TEMs), both of which have been proposed to be platforms for HIV-1 assembly. However, a variety of studies have demonstrated that lipid rafts and TEMs are distinct microdomains in the absence of HIV-1 infection. To measure the impact of Gag on microdomain behaviors, we took advantage of two assays: an antibody-mediated copatching assay and a Förster resonance energy transfer (FRET) assay that measures the clustering of microdomain markers in live cells without antibody-mediated patching. We found that lipid rafts and TEMs copatched and clustered to a greater extent in the presence of membrane-bound Gag in both assays, suggesting that Gag induces the coalescence of lipid rafts and TEMs. Substitutions in membrane binding motifs of Gag revealed that, while Gag membrane binding is necessary to induce coalescence of lipid rafts and TEMs, either acylation of Gag or binding of phosphatidylinositol-(4,5)-bisphosphate is sufficient. Finally, a Gag derivative that is defective in inducing membrane curvature appeared less able to induce lipid raft and TEM coalescence. A higher-resolution analysis of assembly sites by correlative fluorescence and scanning electron microscopy showed that coalescence of clustered lipid rafts and TEMs occurs predominately at completed cell surface virus-like particles, whereas a transmembrane raft marker protein appeared to associate with punctate Gag fluorescence even in the absence of cell surface particles. Together, these results suggest that different membrane microdomain components are recruited in a stepwise manner during assembly.

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“…This FRET-FLIM technique is highly sensitive, and the change in the excited-state lifetime of the donor fluorophore is concentration independent (58) and does not suffer from fluorophore bleed-through. So far, it has been employed in only a few studies of the interaction of viral proteins in live cells (42,47,48). Two-photon-induced FRET-FLIM (2P-FRET-FLIM) provides several advantages over the single-photon method, including reduced phototoxicity (by use of near-infrared excitation light that is not absorbed by cellular components) and reduced bleaching of the fluorophore (10,50,51).…”

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confidence: 99%

Interaction of Poxvirus Intracellular Mature Virion Proteins with the TPR Domain of Kinesin Light Chain in Live Infected Cells Revealed by Two-Photon-Induced Fluorescence Resonance Energy Transfer Fluorescence Lifetime Imaging Microscopy

Jeshtadi

Burgos

Stubbs

et al. 2010

J Virol

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Lateral Distribution of the Transmembrane Domain of Influenza Virus Hemagglutinin Revealed by Time-resolved Fluorescence Imaging

Abstract: Influenza virus hemagglutinin (HA) has been suggested to be enriched in liquid-ordered lipid domains named rafts, which represent an important step in virus assembly. We employed

Cited by 76 publications

References 41 publications

Gel Domains in the Plasma Membrane of Saccharomyces cerevisiae

Gel Domains in the Plasma Membrane of Saccharomyces cerevisiae

Gag Induces the Coalescence of Clustered Lipid Rafts and Tetraspanin-Enriched Microdomains at HIV-1 Assembly Sites on the Plasma Membrane

Interaction of Poxvirus Intracellular Mature Virion Proteins with the TPR Domain of Kinesin Light Chain in Live Infected Cells Revealed by Two-Photon-Induced Fluorescence Resonance Energy Transfer Fluorescence Lifetime Imaging Microscopy

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