Lectin binding patterns to plasmalemmal glycoconjugates of goblet cells undergoing differentiation in vitro.
The plasmalemmal glycoconjugates of the HT29-18N2 (N2) cell line were characterized on cells grown as 1) undifferentiated multilayers in glucose-containing culture media and 2) monolayers of columnar cells acquiring the goblet cell phenotype in glucose-free media. Lectins were unable to bind sheets of detached N2 cells in the absence of fixation. Following fixation with aldehydes, a dramatic unmasking of lectin binding sites was seen. When fixed monolayers were stained prior to embedding, biotinylated lectins, visualized by the avidin-biotin-complexed peroxidase technique, were more efficient than collodial gold-coupled lectins. Lectin binding sites could also be detected by using collodial gold-coupled lectins to stain monolayers embedded in LR White, Lowicryl K4M, and Lowicryl HM20. The binding of 5 lectins (wheat germ, Dolichos bifluros, peanut, soybean, and Ulex europeus) was found to be independent of the stage of differentiation; "pre-differentiated" columnar cells which had prominent microvilli and no or few mucous secretory granules had identical staining patterns as well-differentiated goblet cells with large numbers of secretory granules. Ricinus communis I was the only lectin whose binding was influenced by the stage of differentiation; it intensely labeled undifferentiated multilayers of N2 cells but only weakly labeled basolateral membranes of differentiated monolayers. Canavalia ensiformas (ConA) caused a moderate and even labeling of both apical and basolateral membranes of fixed monolayers stained prior to embedding, but post-embedding labeling revealed heavy labeling along the lateral margins of all columnar cells and weak to moderate binding along the apical and basal cell surface.
Division of Biological Sciences, University of Missouri, Columbia 65211.
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Frisch
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Phillips
D000180:Acrylic Resins; D002455:Cell Division; D002478:Cells, Cultured; D003106:Colon / Q000166:cytology; D005404:Fixatives; D006001:Glycoconjugates / Q000378:metabolism*; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D037102:Lectins / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000180:Acrylic Resins; D005404:Fixatives; D006001:Glycoconjugates; D037102:Lectins; C037883:Lowicryl 4KM resin