scholarly journals The use of lectins and cholera toxin for the detection of surface carbohydrates of cultured neurons and neuroblastoma.

1979 ◽  
Vol 27 (8) ◽  
pp. 1165-1166 ◽  
Author(s):  
N K Gonatas
Keyword(s):  
Horseradish Peroxidase ◽  
Cholera Toxin ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Neuroblastoma Cells ◽  
Cultured Neurons ◽  
Plasma Membrane Receptor ◽  
Surface Carbohydrates ◽  
Adsorptive Endocytosis

Conjugates of horseradish peroxidase with the lectins ricin (d-galactose), wheat germ agglutinin (N-acetylglucosamine), phytohemagglutinin (N-acetylgalactosamine), and with cholera toxin (GM1 ganglioside) were used for a cytochemical detection of corresponding termin al carbohydrates, or glycolipids on cell surfaces of cultured neurons and neuroblastoma cells. Cells were labeled at 4 degrees C with the above ligands and their adsorptive endocytosis was studied after incubations at 37 degrees C in a medium free of ligand. Peroxidase was detected by the method of Graham and Karnovsky (J. Histochem. Cytochem. 14:291, 1966). Lectins and cholera toxin underwent endocytosis in cisternae and vesicles of GERL (Golgi-Endoplasmic Reticulum-Lysosome). We suggest that GERL is the primary ercipieint of adsorptively endocytosed plasma membrane "receptor"-ligand complexes which are thus degraded or possibly reutilized (recycling). Wheat germ agglutinin-horseradish peroxidase conjugates used in vivo for studies of retrograde axonal transport were significantly more sensitive than free horseradish peroxidase.

In vivo uptake of wheat germ agglutinin-horseradish peroxidase conjugates into neuronal gerl and lysosomes

1980 ◽  
Vol 188 (2) ◽  
pp. 465-472 ◽  
Author(s):  
Clive G. Harper ◽  
Jacqueline O. Gonatas ◽  
Anna Stieber ◽  
Nicholas K. Gonatas
Keyword(s):  
Horseradish Peroxidase ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
In Vivo Uptake

scholarly journals Endocytosis of cholera toxin in GERL-like structures of murine neuroblastoma cells pretreated with GM1 ganglioside. Cholera toxin internalization into Neuroblastoma GERL.

1979 ◽  
Vol 81 (3) ◽  
pp. 543-554 ◽  
Author(s):  
K C Joseph ◽  
A Stieber ◽  
N K Gonatas
Keyword(s):  
Golgi Apparatus ◽  
Horseradish Peroxidase ◽  
Cholera Toxin ◽  
Plasma Membranes ◽  
Neuroblastoma Cells ◽  
Gm1 Ganglioside ◽  
Murine Neuroblastoma ◽  
Positive Elements ◽  
Subunit B ◽  
Cytochemical Marker

Cholera toxin (CT), covalently attached to horseradish peroxidase (HRP), is a specific cytochemical marker for GM1 ganglioside (GM1) and retains the ability of the native toxin to raise levels of cyclic AMP in avian erythrocytes. Using a cytochemical stain for HRP, we found that 9% of control cultured murine neuroblastoma cells bound cholera toxin-horseradish peroxidase conjugates (CT-HRP) on their surfaces after incubations for 1 h at 4 degrees C. Exogenous GM1, the natural receptor of CT, becomes associated in the culture medium with the plasma membranes of these cells so that 96% of cells are stained. Cells preincubated with GM1 at 4 degrees C were exposed to CT-HRP for 1 h at 4 degrees C. After washing, cells were incubated at 37 degrees C for 30 min-24 h. Endocytosis of CT-HRP occurred within 30 min and CT-HRP remained, throughout the 24-h period, in tubules, vesicles, and cisternae often found near the Golgi apparatus; this aggregate of peroxidase-positive elements probably corresponds to Golgi apparatus-endoplasmic reticulum-lysosomes (GERL) of neurons. In metaphase cells, CT-HRP was observed in aggregates of vesicles and tubules clustered near the centriole. Conjugates of HRP with subunit B, the GM1 binding component of CT, were internalized by cells pretreated with GM1 as was CT-HRP. The 9% of neuroblastoma cells binding CT-HRP in the absence of exogenous GM1 internalized the ligand in a manner indistinguishable from that of the treated cells. These findings indicate that, in neuroblastoma cells, a system of vesicles, tubules, and cisternae, analogous to GERL of neurons, is the primary recipient of adsorptive endocytosis of CT bound to endogenous or exogenously introduced GM1.

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