GPI-anchored proteins, glycosphingolipids, and sphingomyelin are sequestered to caveolae only after crosslinking.

Fujimoto, Toyoshi

doi:10.1177/44.8.8756764

Cited by 113 publications

(88 citation statements)

References 32 publications

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“…The plasma membrane distribution of CTXB and GPI-anchored proteins was more diffuse and extensive than that of caveolin-1 or -2, which could be detected by indirect immunofluorescence microscopy in HeLa and NRK cells but not in Fao cells (our unpublished results). That neither the GPI-anchored proteins nor CTXB appear to be exclusively associated with caveolin/caveolae is in agreement with previous reports (Montesano et al, 1982;Mayor et al, 1994;Parton, 1994;Fujimoto, 1996). Because clustering of CTXB in caveolae in living cells is enhanced with increasing the temperature of incubation (Parton, 1994), in our experiments cells were labeled on ice, a condition in which minimal clustering occurs, and then immediately fixed.…”

Section: Fret Microscopy Of Lipid Raft Componentssupporting

confidence: 93%

“…If cells were instead labeled with monoclonal antibodies followed by anti-mouse Ig and briefly incubated at 37°C before fixation, discrete spots of label were seen (our unpublished results). These puncta were similar to those reported in previous studies (Rothberg et al, 1990;Mayor et al, 1994;Fujimoto, 1996;Harder et al, 1998;Kenworthy and Edidin, 1998). The plasma membrane distribution of CTXB and GPI-anchored proteins was more diffuse and extensive than that of caveolin-1 or -2, which could be detected by indirect immunofluorescence microscopy in HeLa and NRK cells but not in Fao cells (our unpublished results).…”

Section: Fret Microscopy Of Lipid Raft Componentssupporting

confidence: 92%

“…Such domains containing clustered GPI-anchored proteins can sometimes be detected by conventional light or electron microscopy (Matsuura et al 1984;Latker et al 1987;Kobayashi and Robinson, 1991;van den Berg et al, 1995) (reviewed in Anderson [1998]). In other cases clusters of GPI-anchored proteins or other lipid raft components are only apparent when they have been crosslinked with secondary antibodies (Howell et al 1987;Rothberg et al, 1990;Fra et al, 1994;Mayor et al, 1994;Parton et al, 1994;Fujimoto, 1996;Harder et al, 1998). This suggests that in vivo, lipid rafts may be small or dynamic structures that are aggregated and stabilized by detergent extraction or by antibody-induced cross-linking (Edidin, 1997;Harder and Simons, 1997;Simons and Ikonen, 1997;Weimbs et al, 1997;Brown and London, 1998;Jacobson and Dietrich, 1999).…”

Section: Introductionmentioning

confidence: 99%

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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: Fret Microscopy Of Lipid Raft Componentssupporting

confidence: 93%

Section: Fret Microscopy Of Lipid Raft Componentssupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

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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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“…Others, however, claim that GPIanchored proteins aggregate only after addition of detergents or antibodies [12, 13,15,25]. We investigated the connection between our crosslinked oligomers and the clusters found after detergent extraction of cells by crosslinking MDCK GH-DAF cells after extraction with Triton-X-114 detergent using mild or harsh conditions (see Methods; the latter is commonly used for preparing detergent-insoluble complexes).…”

mentioning

confidence: 99%

“…For instance, the discovery of caveolin-1, a protein of the caveolar coat, in detergent extracts enriched in glycosphingolipids, cholesterol and GPI-anchored proteins has led to the proposed existence of rafts containing caveolin-1 and GPI-anchored proteins [10]. However, there is accumulating evidence that there is no enrichment of GPI-anchored proteins in caveolae [25,28,29]; the same is true for trimeric G proteins, which are also found in a buoyant raft fraction but are neither functionally nor morphologically connected with caveolin [30]. The rules governing the interplay between the inclusion and exclusion of proteins into rafts remain to be defined.…”

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confidence: 99%

Microdomains of GPI-anchored proteins in living cells revealed by crosslinking

Friedrichson

Kurzchalia

1998

Nature

521

343

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| There is some discussion as to whether glycosyl-phosphatidylinositol(GPI)-anchored proteins occur in microdomains in the cell membrane. These putative microdomains have been implicated in processes such as sorting in polarized cells and signal transduction. Complexes enriched in GPI-anchored proteins, cholesterol and glycosphingolipids have been isolated from cell membranes by using non-ionic detergents: these complexes were thought to represent a clustered arrangement of GPI-anchored proteins. However, results obtained when clustering of GPI-anchored proteins induced by antibodies or by detergents was prevented support the idea of a dispersed surface distribution of GPI-anchored proteins at steady state. Here we use chemical crosslinking to show that membrane microdomains of a GPI-anchored protein exist at the surface in living cells. This clustering is specific for the GPIanchored form, as two transmembrane forms bearing the same ectodomain do not form oligomers. Depletion of membrane cholesterol causes the clustering of GPI-anchored proteins to break up, whereas treatment of cells with detergent substantially increases the size of the complexes. We find that in living cells these GPI-anchored proteins reside in microdomains consisting of at least 15 molecules, which are much smaller than those seen after detergent extraction.To study the association of GPI-anchored proteins on the cell surface of intact cells, we used growth hormone (GH) with the GPI-anchoring signal from decay-accelerating factor (DAF) attached to its carboxy terminus (GH-DAF) (Fig.1a). This molecule is an established model for a GPIanchored protein [14]. When MDCK cells that permanently express GH-DAF (MDCK GH-DAF) were chemically crosslinked at 4 °C with the membraneimpermeable agent bis(sulphosuccinimidyl)suberate (BS3), a prominent band corresponding to a relative molecular mass of 46K (dimer), and a smear from ≈60K to ≈300K were detected, suggesting the existence of microdomains. The crosslinking efficiency for 0.5 mM BS3 was 71%. When crosslinked at 37 °C, the pattern was similar (crosslinking efficiency was 86%), indicating that microdomains also exist at physiological temperatures. Increasing concentrations of BS3 (4 °C) or increasing the incubation time with crosslinker (37 °C) shifted the pattern of crosslinked products to high-molecular-mass forms (Fig.1b). To analyse the oligomers formed, we used the cleavable BS3 analogue 3,3'-dithiobis-(sulphosuccinimidylpropionate) (DTSSP) and twodimensional (2D) electrophoresis. Electrophoresis in the first dimension gave a crosslinking pattern similar to that obtained with BS3, whereas the second dimension under reducing conditions revealed that the crosslinked oligomers consisted primarily of GH-DAF (arrow in Fig.1c); some minor interaction partners were also detected (arrowheads in Fig.1c). Because the length of the spacer arm of BS3 is only 1.14 nm, the detection of crosslinked GH-DAF oligomers indicates that these molecules are close together on the cell surface at steady stat...

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Targeted Transfer of Polyethylenimine–Avidin–DNA Bioconjugates to Hematopoietic Cells Using Biotinylated Monoclonal Antibodies

Wojda

Miller

2000

Journal of Pharmaceutical Sciences

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GPI-anchored proteins, glycosphingolipids, and sphingomyelin are sequestered to caveolae only after crosslinking.

Cited by 113 publications

References 32 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

Microdomains of GPI-anchored proteins in living cells revealed by crosslinking

Targeted Transfer of Polyethylenimine–Avidin–DNA Bioconjugates to Hematopoietic Cells Using Biotinylated Monoclonal Antibodies

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