Intracellular traffic of lipid raft by lipid-specific probe

本研究的目的是利用脂质特异性探针研究脂筏的细胞内运输。在支持期间取得了以下成果。1.脂质特异性探针的特异性分析：将活培养的细胞与荧光PEG-胆固醇（fPEG-Chol）或无毒的胞溶素（Lys）孵育，并在共聚焦激光扫描显微镜下观察。fPEG-Chol与霍乱毒素共定位，但Lys不与霍乱毒素共定位。2. PEG-胆固醇在红细胞中的分布：PEG-胆固醇诱导活红细胞胞吐。采用速冻深蚀电镜观察细胞形态。PEG-Chol分子在棘突处聚集，表明PEG-Chol的聚集引起红细胞膜的挤出。3.脂筏组分的内吞作用：通过电子显微镜分析F592成纤维细胞中胞溶素的摄取。胞溶素孵育5分钟后未被内吞。然而，30分钟后，胞溶素被内吞在晚期核内体中。4.神经鞘磷脂在细胞表面的二维分布：将神经鞘磷脂的表面分布与神经节苷脂GM 1的表面分布进行比较。用溶素和霍乱毒素以及金标记的二抗标记后，在Formvar包被的网格上撕下细胞膜的片段。两种探针形成微区，但它们不是共定位的。这一结果通过使用Ripley’s K-函数检验的图像分析得到证实。这些结果表明了脂筏的多样性。5.通过胞溶素-金的鞘磷脂的细胞内运输：为了避免探针聚集，将胞溶素直接与胶体金缀合。即使没有探针的聚集，鞘氨醇在10分钟内也没有被内吞。

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.