Selective anchoring in the specific plasma membrane domain: a role in epithelial cell polarity

FRAP reports on the diffusion coefficient, D, of a labeled molecule of interest, on the fraction of label in a region of the cell that can diffuse at all, and on the fraction of cells in a population in which no diffusion can be measured (hereafter referred to as totally immobile fraction [TIF]). We used FRAP to determine all of these parameters for fluorescent Fab fragments of A2 and B1 mAbs. We also labeled the class I major histocompatibility complex antigens, DLA, with fluorescent B2-m, a low molecular mass (~12 kD) monovalent probe. No active Fab fragments of A1 mAb (an IgM) or B2 mAb could be obtained. The measurements were carried out on MDCK monolayers grown on nylon filter chambers similar to those described by Cereijido et al. (12) , except that the filters were covered by native, instead of fixed, collagen. Fluorescent Fab fragments or B2-m were added to either the apical or the basal side of the monolayers to study the mobility of the antigen in the apical and in the basolateral membrane. Comparative measurements of A2 in both membranes were, unfortunately, not possible, because this antigen is so intensely polarized that no specific signal can be detected on the basal membrane. The results are shown in Table IV .