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.
Frisch
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Division of Biological Sciences, University of Missouri, Columbia 65211.
Phillips
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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