| 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...

Exogenous application of gangliosides to cells affects many cellular functions. We asked whether these effects could be attributed to the influence of gangliosides on the properties of sphingolipid-cholesterol microdomains on the plasma membrane, also termed rafts. The latter are envisaged as lateral assemblies of sphingolipids (including gangliosides), cholesterol, and a specific set of proteins. Rafts have been implicated in processes such as membrane trafficking, signal transduction, and cell adhesion. Recently, using a chemical cross-linking approach with Madin-Darby canine kidney (MDCK) cells permanently expressing a GPI-anchored form of growth hormone decay accelerating factor (GH-DAF) as a model system, we could show that GPI-anchored proteins are clustered in rafts in living cells. Moreover, this clustering was dependent on the level of cholesterol in the cell. Here we show that incubation of MDCK cells with gangliosides abolished subsequent chemical cross-linking of GH-DAF. Furthermore, insertion of gangliosides into the plasma membrane of MDCK GH-DAF cells renders GH-DAF soluble when subjected to extraction with Triton X-114 at 4 degrees C. Our data suggest that exogenous application of gangliosides displaces GPI-anchored proteins from sphingolipid-cholesterol microdomains in living cells.

This study was undertaken to investigate the distribution of vitamin E in the macular and peripheral regions of the human retina, retinal pigment epithelium (RPE) and choroid as a function of age. High-performance liquid chromatography (HPLC) was used to measure alpha- and gamma-tocopherol levels quantitatively using tocol as an internal standard. In 57 out of 70 donor eyes (ages 9-104) the macular region was isolated and the tocopherols analyzed. The conventional brush method and a new vortex method were used to isolate the retinal pigment epithelium cells. Similar trends for the vitamin E levels (increase to the 5th decade, decrease after 7th decade) were found for the macular and peripheral retina and for the macular RPE. In the peripheral RPE a slight continuous increase with age was found. The vitamin E levels are higher in the RPE than in the retina, for both macular and peripheral regions. The amounts of vitamin E/mg protein are lower in the macular retina than in the peripheral retina, whereas in the RPE there is no difference in vitamin E content between macular and peripheral regions. A simple method based on a gentle vortex step was found to offer several advantages over the more generally used isolation of RPE cells based on brushing, and there was no difference in recovery of vitamin E in RPE cells when they were isolated by either isolation technique. It was also found that denominators, used to express the values of vitamin E in tissues should, be used with care since age dependent trends in parameters/denominators could be caused by trends in the denominators only.

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