Specific lectins map the distribution of fibronectin and beta 1-integrin on living MDCK cells

Abstract

The expression and dynamics of bound fibronectin and the sialylated integral membrane protein, beta 1-integrin, were analyzed on the apical membrane of living MDCK cells. Fibronectin was identified by its specific binding of fluorescent peanut agglutinin and sialylated beta 1-integrin by its binding of Sambucus nigra agglutinin. Confocal epifluorescence microscopy and laser scanning cytometry determined the distribution and abundance of binding sites of the two fluorescently labeled lectins. Both fibronectin and beta 1-integrin were restricted to specific regions uniformly distributed over the entire apical surface. Apical-surface fibronectin binding varied much more between cells than did the expression of beta 1-integrin. Sialylated beta 1-integrin colocalized >92% with membrane microplicae while fibronectin was unrelated to these surface structures. This lack of colocalization of the proteins was confirmed by double-labeling experiments. From the maturation dependence of the fibronectin-binding capacity and the differences in protein turnover times, it was evident that fibronectin did not bind to sialylated beta 1-integrin. Furthermore, desialylation of beta 1-integrin uncovered additional fibronectin receptors on the apical membrane. We conclude that these lectins permit tracking of two membrane-associated glycoproteins in living cells and that fibronectin binds only to desialylated beta 1-integrin on MDCK cells.