21 June 2004 FRET reveals the organization of different receptor-ligand complexes (polymeric IgA-R and Transferrin-R) in endocytic membranes of polarized MDCK cells
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Abstract
FRET-based assay has been used to determine the organization of transferrin-receptor bound to holo-transferrin in basolateral endocytic membranes and compare it to the previously characterized clustered organization of polymeric IgA-receptor (pIgA-R) bound to pIgA-R ligand in apical endocytic membranes. In polarized MDCK-PTR cells, we have internalized holo-transferrin from the basolateral plasma membrane - labeled with donor and acceptor fluorophores. Transferrin-receptor-holo-transferrin complexes were imaged in the basolateral endocytic compartment using FRET confocal laser scanning microscopy in fixed and live MDCK polarized cells. A two-parameter FRET assay demonstrates whether complexes are randomly distributed or clustered: Acceptor's positive impact on E% signifies random distribution; E% being independent of acceptor fluorescence levels indicates clusters. A second parameter for clustering is E% being negatively dependent on D:A ratios. Our results indicating a clustered organization of transferrin-receptor-holo transferrin complexes fit the well-known homodimeric structure of transferrin-receptor.
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Horst K. Wallrabe, Margarida Barroso, "FRET reveals the organization of different receptor-ligand complexes (polymeric IgA-R and Transferrin-R) in endocytic membranes of polarized MDCK cells", Proc. SPIE 5323, Multiphoton Microscopy in the Biomedical Sciences IV, (21 June 2004); doi: 10.1117/12.539755; https://doi.org/10.1117/12.539755
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