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This Article Full Text Full Text (PDF) Supplemental Movie All Versions of this Article: 293/5/C1717    most recent 00309.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Subramanian, V. S. Articles by Said, H. M. Search for Related Content PubMed PubMed Citation Articles by Subramanian, V. S. Articles by Said, H. M.

PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Tight junction targeting and intracellular trafficking of occludin in polarized epithelial cells

Departments of ¹Medicine and ²Physiology and Biophysics, University of California, Irvine, California; ³Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota; ⁴Department of Internal Medicine, University of New Mexico, Albuquerque, New Mexico; and ⁵Department of Veterans Affairs Medical Center, Long Beach, California

Submitted 19 July 2007 ; accepted in final form 4 September 2007

Occludin, a transmembrane (TM)-spanning protein, is an integral component of the tight junctional (TJ) complexes that regulate epithelial integrity and paracellular barrier function. However, the molecular determinants that dictate occludin targeting and delivery to the TJs remain unclear. Here, using live cell imaging of yellow fluorescent protein-labeled occludin fragments, we resolved the intracellular trafficking of occludin-fusion proteins in polarized Madin-Darby canine kidney and Caco-2 cells to delineate the regions within the occludin polypeptide that are important for occludin targeting to the TJs. Live cell confocal imaging showed that complete or partial truncation of the COOH-terminal tail of the occludin polypeptide did not prevent occludin targeting to the TJs in epithelial cell lines. Progressive truncations into the COOH-terminal tail decreased the efficiency of occludin expression; after the removal of the regions proximal to the fourth transmembrane domain (TM4), the efficiency of expression increased. However, further deletions into the TM4 abolished TJ targeting, which resulted in constructs that were retained intracellularly within the endoplasmic reticulum. The full-length occludin polypeptide trafficked to the cell surface within a heterogenous population of intracellular vesicles that delivered occludin to the plasma membrane in a microtubule- and temperature-dependent manner. In contrast, the steady-state localization of occludin at the cell surface was dependent on intact microfilaments but not microtubules.

epithelium

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