UBC Theses and Dissertations
Visualization and characterization of cell surface glycoproteins on mouse neuroblastoma cells Maher, Pamela A.
The cell surface glycoproteins of mouse neuroblastoma cells were visualized using fluorescent lectins in combination with fluorescent light microscopy and SDS-gel electrophoresis, lectin-microsphere conjugates in combination with scanning electron microscopy, and iodinated lectins. The lectins used in these studies were: Con-canavalin A (ConA) which is specific for α-D-mannose and α-D-glucose groups, wheat germ agglutinin (WGA) which binds to N-acetylglucosa-mine oligomers and nicinus communis agglutinin (RCAI) which is specific for D-galactose residues. All three lectins were found to have over 10⁷ high affinity (K[sub d]= 2 x 10⁻⁷ M) binding sites on the neuroblastoma cell surface. These sites were found to be densely and randomly distributed over the surfaces of cells which were fixed with glutaraldehyde before labelling or labeled at 4°C. However, when the cells were labeled at 23°C or 37°C, the lectin receptors were found to undergo redistribution. All three lectins were internalized by the cells in an energy- dependent process when the cells were treated with the lectins for 60 min or more at 37°C. However, the patterns of redistribution for the different lectin receptors were not the same. When cells were labeled with ConA, the receptors were shown to patch and then clear from the surface of the cells. Once the label had completely cleared from the cell surface, the cells could not be labeled with additional ConA, even when the first labelling was done using ConA at concentrations well below saturation. However, when cells were treated in the same way with WGA or RCAI, a heavy, uniform display of marker was seen on the cell surface at all times. It was only when the cells were briefly labeled with these lectins and then incubated in buffer that it became apparent that these receptors could also patch and clear from the cell surface. In addition, in order to clear all the receptors for these two lectins from the cell surface, it was necessary to label the cells with saturating concentrations of lectin. Thus, labeled and unlabeled ConA receptors on the neuroblastoma cells undergo co-ordinate redistribution whereas labeled WGA and RCAI receptors redistribute independently of their unlabeled counterparts. Studies with drugs which disrupt the various cytoskeletal elements suggested that the microfilament system played a role in the coordinate redistribution of ConA receptors. Double labelling studies with both different iodinated and fluorescent lectins indicated that the binding sites for WGA were not associated with those for ConA. WGA binding sites did appear to be directly associated with many of the binding sites for RCAI. Studies with ConA and RCAI were not carried out because it was shown that ConA binds to RCAI. Using the fluorescent lectins in combination with SDS-gel electrophoresis, the lectin-binding polypeptides from the neuroblastoma cell membranes were identified and characterized. Plasma membranes were purified from the neuroblastoma cells using hypotonic disruption followed by differential and isopycnic gradient centifugation. The membrane preparation showed a 10-fold increase in the specific activity of two plasma membrane markers and little contamination by other cell components. The membranes were dissociated in SDS and run on SDS-polyacrylamide gels to separate out the different polypeptides. The gels were fixed and stained with fluorescent lectins. WGA and RCAI were both found to bind almost exclusively to a single polypeptide with a molecular weight of 30,000 daltons. This polypeptide also stained strongly for carbohydrate after periodate oxidation. ConA, on the other hand, bound to over 20 polypeptides, most of which had molecular weights between 60,000 and 120,000 daltons. However, when the cells were made to internalize all their ConA receptors and their membranes isolated and run on gels, four of the ConA-staining polypeptides were found to be absent as compared to membranes from untreated cells. Thus, it appears that only four of the ConA-binding polypeptides seen on the gels were available for ConA binding at the cell sur- face. These polypeptides also labeled with I¹²⁵ when intact cells were treated with I¹²⁵ and lactoperoxidase.
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