MEMBRANE G_(M1) GANGLIOSIDE MEDIATED ENDOCYTOSIS OF CHOLERA TOXIN-HORSERADDISH PEROXIDASE COMPLEX (CT-HRP) IN CULTURED NEURONS
Wu Gu-sheng and Pao Xuan (Shanghai Brain Research Institute,Academia Sinica)
In this experiment,cells dissociated fromnewborn rats cerebellum and cultured in vitrofor 7—14 days were exposed to cholera toxin-borseradish peroxidase complex (CT-HRP)for 0.5—1 h.at 4℃.After rinsing throu-ghly,cells were incubated at 36.5℃ for 15min—3h.topermite endocytosis of boundtoxin on membrane.Finally an electro mi-croscopical cytocbemical reaction for HRPwas achieved. It was found that,at 4℃,CT-HRPbound on membrane surface of neurons andsome oligodendrocytes,This suggests that G_(M1)distributes on the outlayer of cell membrane.According to the density of reaction granulesof CT-HRP under electromicroscopy,it wasshown that the concentration of G_(M1) on neuronalmembrane was higher than on oligodendrocyte. The endocytosis of bound CT-HRP intoneuronal cytoplasm was given rise within 15min.after being incubated at 36.5℃.Suchendocytosis,termed as receptors G_(M1) mediatedendocytosis (RME),proceed frequently ondendrites as well as on cell bodies.Comparedwith endocytosis of free HRP into neuronsin which endovesicles containing free HRPwere fused into lysosomes to be degradated,the endovesicles containing G_(M1)-CT-HRPcomplex found their ways primary to Golgiapparatus-Endoplasmic Reticulum-lysosomessystem (GERL) which is functionally related tomembrane recycle.If cells were incubated for2—3 h.longer,the amount of CT-HRP boundon neuronal membrane did not change signi-ficantly:lots of positive vesicles still aggre-gated in GERL as before:and many otherspresent themselves in cytoplasm near the mem-brane and along the microtubes. Most of all CT-HRP bound on oligoden-drocyte membrane was internalized withinincubating of 15 min.A large number of en-dovesicles diffused in cytoplasm.As the timeof incubation lasted,the positive vesicles fu-sed into lysosome. The possible causes of the different waysof CT-HRP endocytosis in neurons and oli-godendrocytes and their biological significancewere discussed.