Intracellular traffic of lipid raft by lipid-specific probe

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The purpose of this study was to investigate the intracellular traffic of lipid raft by lipid-specific probes. The following results were obtained during the supported period. 1.Analysis of specificity of lipid-specific probes : Live cultured cells were incubated with fluorescent PEG-cholesterol (fPEG-Chol) or non-toxic lysenin (Lys), and observed in a confocal laser-scanning microscope. fPEG-Chol was colocalized with cholera toxin, but Lys was not. 2.Distribution of PEG-cholesterol in erythrocyte : PEG-Chol induced echiocytosis of live erythrocytes. The cells were observed by quick-freeze, deep-etch electron microscopy. PEG-Chol molecules were aggregated at echinocytic process, suggesting that accumulation of PEG-Choi induced extrusion of erythrocyte membrane. 3.Endocytosis of lipid raft components : Uptake of lysenin in F592 fibroblasts were analyzed by an electron microscope. Lysenin was not endocytosed after incubating for 5 min. However, after 30 min, lysenin was endocytosed in late endosomes. 4.2D-distribution of sphingomyelin on cell surface : Surface distribution of sphingomyelin was compared to that of ganglioside GM1. After labeling with lysenin and cholera toxin, and gold-labeled secondary antibody, fragments of cell membranes were ripped-off on Formvar-coated grids. Both probes formed microdomain, but they were not co-localized. This result was confirmed by image analysis using Ripley's K-function test. These results indicated the diversity of lipid rafts. 5.Intracellular traffic of sphingomyelin by lysenin-gold : To avoid aggregation of probe, lysenin was directly conjugated with colloidal gold. Even without aggregation of probe, sphingomylin was not endocytosed within 10 min.