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Vol. 18, Issue 6, 2226-2243, June 2007

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Receptor Complexes Cotransported via Polarized Endocytic Pathways Form Clusters with Distinct Organizations

H. Wallrabe^*,, G. Bonamy^,,, A. Periasamy^*, and M. Barroso^||

*Department of Biology, W. M. Keck Center for Cellular Imaging, University of Virginia, Charlottesville, VA 22904; Hudson Alpha Institute for Biotechnology, Huntsville, AL 35801; and ^||Center for Cardiovascular Sciences, Albany Medical College, Albany, NY 12208

Submitted August 11, 2006; Revised February 8, 2007; Accepted March 26, 2007
Monitoring Editor: Francis Barr

Previously, FRET confocal microscopy has shown that polymeric IgA-receptor (pIgA-R) is distributed in a clustered manner in apical endosomes. To test whether different membrane-bound components form clusters during membrane trafficking, live-cell quantitative FRET was used to characterize the organization of pIgA-R and transferrin receptor (TFR) in endocytic membranes of polarized MDCK cells upon internalization of donor- and acceptor-labeled ligands. We show that pIgA-R and TFR complexes form increasingly organized clusters during cotransport from basolateral to perinuclear endosomes. The organization of these receptor clusters in basolateral versus perinuclear/apical endosomes is significantly different; the former showing a mixed random/clustered distribution while the latter highly organized clusters. Our results indicate that although both perinuclear and apical endosomes comprise pIgA-R and TFR clusters, their E% levels are significantly different suggesting that these receptors are packed into clusters in a distinct manner. The quantitative FRET-based assay presented here suggests that different receptor complexes form clusters, with diverse levels of organization, while being cotransported via the polarized endocytic pathways.

These authors contributed equally to this work.

Present address: Department of Functional Genomics, Genomic Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121.

Address correspondence to: Margarida Barroso (barrosm{at}mail.amc.edu)

Abbreviations used: A, acceptor intensity levels; D, unquenched donor intensity levels; D/A, unquenched donor/acceptor ratios; FRET, Förster resonance energy transfer; pIgA-R, polymeric IgA-receptor; TFR, transferrin-receptor; Tfn, holo-transferrin; pIgA-R complexes, pIgA-R-pIgA-R ligand complexes; TFR complexes, TFR-Tfn complexes.