Exogenous Administration of Gangliosides Displaces GPI-anchored Proteins from Lipid Microdomains in Living Cells

Simons, Mikael; Friedrichson, Tim; Schulz, Jörg B.; Pitto, Marina; Masserini, Massimo; Kurzchalia, Teymuras V.

doi:10.1091/mbc.10.10.3187

Cited by 93 publications

(77 citation statements)

References 41 publications

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“…Although clustering of lipid raft components can be disrupted by altering levels of various raft components (Varma and Mayor, 1998;Simons et al, 1999), it seems unlikely that such an effect occurs in our experiments, which were performed using endogenous markers for rafts. We obtained a similar, density-dependent energy transfer for both endogenous (this study) and transfected (Kenworthy and Edidin, 1998) GPI-anchored proteins, indicating that these molecules had not been artificially dispersed from a clustered distribution because of saturation of the available raft lipids or competition for enrichment in a limited number of clusters.…”

Section: Discussionmentioning

confidence: 92%

High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

Kenworthy

Petranova

Edidin

2000

MBoC

406

311

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“Lipid rafts” enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes. However, evidence supporting their existence has been indirect and controversial. In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface. The results of these studies, each based on a single protein, gave conflicting views of rafts. To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types. FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane. However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains. We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface.

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Section: Discussionmentioning

confidence: 92%

High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

Kenworthy

Petranova

Edidin

2000

MBoC

406

311

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“…It is also possible that GM1 alters raft-like domain structure, reducing Thy-1 partitioning into them. Indeed, a recent study has shown that increased levels of GM1 displace GPI-anchored proteins from sphingolipidcholesterol microdomains in living cells (48).…”

Section: Discussionmentioning

confidence: 99%

Partitioning of Thy-1, GM1, and cross-linked phospholipid analogs into lipid rafts reconstituted in supported model membrane monolayers

Dietrich

Volovyk

Levi

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

317

257

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As shown earlier, raft-like domains resembling those thought to be present in natural cell membranes can be formed in supported planar lipid monolayers. These liquid-ordered domains coexist with a liquid-disordered phase and form in monolayers prepared both from synthetic lipid mixtures and lipid extracts of the brush border membrane of mouse kidney cells. The domains are detergent-resistant and are highly enriched in the glycosphingolipid GM1. In this work, the properties of these raft-like domains are further explored and compared with properties thought to be central to raft function in plasma membranes. First, it is shown that domain formation and disruption critically depends on the cholesterol density and can be controlled reversibly by treating the monolayers with the cholesterol-sequestering reagent methyl-␤-cyclodextrin. Second, the glycosylphosphatidylinositol-anchored cell-surface protein Thy-1 significantly partitions into the raft-like domains. The extent of this partitioning is reduced when the monolayers contain GM1, indicating that different molecules can compete for domain occupation. Third, the partitioning of a saturated phospholipid analog into the raft phase is dramatically increased (15% to 65%) after cross-linking with antibodies, whereas the distribution of a doubly unsaturated phospholipid analog is not significantly affected by cross-linking (Ϸ10%). This result demonstrates that cross-linking, a process known to be important for certain cell-signaling processes, can selectively translocate molecules to liquid-ordered domains.membrane domains ͉ receptor cross-linking ͉ cholesterol ͉ glycosylphosphatidylinositol-anchored proteins ͉ signal transduction T reatment of cell lysates with cold nonionic detergent and subsequent sucrose gradient centrifugation allows extraction of a detergent-resistant membrane fraction (DRM) (1, 2). The finding that this fraction is enriched in cholesterol and sphingolipids that form a liquid-ordered phase (3, 4) has led to the hypothesis that the DRM arises directly from discrete liquidordered domains in the plasma membrane termed lipid rafts (5, 6). Such domains could be important for membrane trafficking and sorting (1, 7). Moreover, cell signaling molecules are enriched in the DRM, and the partitioning of these molecules into the DRM is altered during signaling (8-12). Extraction of cholesterol from plasma membranes affects the abundance of molecules found in the DRM and impairs processes like membrane trafficking (13, 14) and cell activation (15, 16). These findings suggest that lipid rafts on plasma membranes are maintained by proper cholesterol levels as functional, preassembled signal transduction complexes (17)(18)(19).Although there is accumulating evidence suggesting that some form of lipid domains or clusters are present in cell membranes, there is still considerable controversy surrounding the basic physical properties of these domains (compositional diversity, size, structure, and dynamics) and their relation to the DRM (for review see refs. 20 and...

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“…This suggests that carbohydrate moiety of GSLs can affect the interaction of TAG-1 with lipid rafts or the physical properties of lipid rafts. A recent report showed that exogenously administered ganglioside, which was incorporated in lipid raft fraction, displaced GPI-anchored proteins from lipid rafts and increased their detergent solubility (77,78). This finding suggests that GSL can affect distribution of GPI-anchored proteins in lipid rafts.…”

Section: Role Of Gsl In Gpi-anchored Protein Signaling In Lipidmentioning

confidence: 95%

Involvement of Gangliosides in Glycosylphosphatidylinositol-anchored Neuronal Cell Adhesion Molecule TAG-1 Signaling in Lipid Rafts

Kasahara¹,

Watanabe²,

Takeuchi³

et al. 2000

Journal of Biological Chemistry

144

129

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The association of ganglioside GD3 with TAG-1, a glycosylphosphatidylinositol-anchored neuronal cell adhesion molecule, was examined by coimmunoprecipitation experiments. Previously, we have shown that the anti-ganglioside GD3 antibody (R24) immunoprecipitated the Src family kinase Lyn from the rat cerebellum, and R24 treatment of primary cerebellar cultures induced Lyn activation and rapid tyrosine phosphorylation of an 80-kDa protein (p80). We now report that R24 coimmunoprecipitates a 135-kDa protein (p135) from primary cerebellar cultures. Treatment with phosphatidylinositol-specific phospholipase C revealed that p135 was glycosylphosphatidylinositol-anchored to the membrane. It was identified as TAG-1 by sequential immunoprecipitation with an anti-TAG-1 antibody. Antibody-mediated cross-linking of TAG-1 induced Lyn activation and rapid tyrosine phosphorylation of p80. Selective inhibitor for Src family kinases reduced the tyrosine phosphorylation of p80. Sucrose density gradient analysis revealed that the TAG-1 and tyrosine-phosphorylated p80 in cerebellar cultures were present in the lipid raft fraction. These data show that TAG-1 transduces signals via Lyn to p80 in the lipid rafts of the cerebellum. Furthermore, degradation of cell-surface glycosphingolipids by endoglycoceramidase induced an alteration of TAG-1 distribution on an OptiPrep gradient and reduced the TAG-1-mediated Lyn activation and tyrosine phosphorylation of p80. These observations suggest that glycosphingolipids are involved in TAG-1-mediated signaling in lipid rafts.Gangliosides, sialic acid-containing glycosphingolipids (GSLs), 1 are found in the outer leaflet of the plasma membrane of all vertebrate cells and are thought to play functional roles in cellular interactions and the control of cell proliferation (1-4).In the nervous system, where gangliosides are particularly abundant, the species and amounts of gangliosides undergo profound changes during development, suggesting that they may play fundamental roles in this process (5). The accumulation of gangliosides within the neurons in ganglioside storage diseases results in extensive neurite outgrowth (6). Exogenously administered gangliosides accelerate the regeneration of neurons in the central nervous system in vivo after lesioning (7). The addition of exogenous gangliosides to primary cultures of neurons and neuroblastoma cells in vitro stimulates cellular differentiation with concomitant neurite sprouting and extension (8 -10). Glucosylceramide synthesis, the first glycosylation step of GSL synthesis, is required for axonal growth in hippocampal neurons (11) and for embryonic development (12). Transfection of the ganglioside GD3 2 synthase cDNA into neuroblastoma cells induces their cholinergic differentiation and neurite sprouting (13). Finally, mice lacking complex gangliosides exhibit axonal degeneration (14). These data show that gangliosides are involved in neural cell differentiation and brain development. However, the molecular mechanisms underlying the ganglioside-depend...

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Exogenous Administration of Gangliosides Displaces GPI-anchored Proteins from Lipid Microdomains in Living Cells

Cited by 93 publications

References 41 publications

High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

Partitioning of Thy-1, GM1, and cross-linked phospholipid analogs into lipid rafts reconstituted in supported model membrane monolayers

Involvement of Gangliosides in Glycosylphosphatidylinositol-anchored Neuronal Cell Adhesion Molecule TAG-1 Signaling in Lipid Rafts

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