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- W2045449764 abstract "Cell membranes are believed to contain lipid rafts which are ordered membrane domains rich in sphingolipids and sterols. Many studies investigating the functional role of lipid rafts are performed by removal of cholesterol from plasma membranes. However, it is uncertain whether the functional changes observed are due to altered raft levels or a more specific cholesterol-dependent effect. To distinguish these cases, we carried out sterol substitution experiments using methyl-β-cyclodextrin with a variety of raft formation-stabilizing and raft formation-destabilizing sterols. This approach was then applied to antibody-induced placental alkaline phosphatase (PLAP)-endocytosis. This process is cholesterol-dependent, but the mechanism of the cholesterol dependence is not known. Sterol depletion and substitution was carried out in MDA-MB-231 cells stably transfected with PLAP. After substitution, cholesterol-depletion and sterol-substitution were confirmed by HP-TLC. To define the effect of sterols on membrane structure TMADPH was added to the cells and fluorescence anisotropy measured. TMADPH anisotropy reflects plasma membrane lipid packing, and high values should be correlated with higher raft levels. Compared to untreated cells, cholesterol-depletion decreased anisotropy as expected. Subsequent replacement of cholesterol or dihydrocholesterol increased anisotropy to a similar or slightly higher level than that prior to depletion, consistent with restoration of raft levels, while anisotropy was somewhat lower with epicholesterol and much lower with lanosterol. Next, antibody-induced PLAP endocytosis was visualized by immunofluorescence. Only substitution with cholesterol restored PLAP internalization levels to an extent similar to untreated control. Decreasing cholesterol or substitution of cholesterol with other sterols inhibited internalization. This suggests that specific details of sterol structure are likely to be more important than tightness of lipid packing or raft levels in plasma membrane for antibody-induced PLAP endocytosis." @default.
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- W2045449764 date "2015-01-01" @default.
- W2045449764 modified "2023-09-29" @default.
- W2045449764 title "Antibody Induced PLAP Endocytosis is Dependent on the Structure and Amount of Sterols in Cellular Plasma Membrane" @default.
- W2045449764 doi "https://doi.org/10.1016/j.bpj.2014.11.573" @default.
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