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FRET microscopy reveals clustered distribution of co-internalized receptor-ligand complexes in the apical recycling endosome of polarized epithelial MDCK cells

Our objective is to study the organization of the apical recycling endosome (ARE) in polarized epithelial MDCK cells. In MDCK cells, stably transfected with polymeric IgA receptor (pIgA-R), anti-pIgA-R [Fab2] ligands labeled with distinct fluorophores were internalized from opposite PM domains at 17 degree(s)C. Internalization at 17 degree(s)C allows the accumulation of basolaterally and apically internalized pIgA-R ligands in the apical recycling compartment. Fluorescence resonance energy transfer (FRET) confocal microscopy analysis was used to determine whether the receptor-ligand complexes are arranged in sub-pixel endosomal cluster domains. Quantitative analysis of FRET efficiency (E%) was carried out using a custom algorithm that removes contamination caused by acceptor excitation by the donor wavelength and by the crosstalk between donor and acceptor emissions in the acceptor channel. The algorithm allows the determination of the unquenched donor levels and thereby the calculation of E%. Average E% is ~42% and it is independent of acceptor levels. Since a random distribution of receptor-ligand complexes would result in E% increasing with rising acceptor levels, our results indicate a cluster distribution of receptor-ligand complexes in the ARE of polarized MDCK cells.