Tight junction targeting and intracellular trafficking of occludin in polarized epithelial cells.
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.
Subramanian
Veedamali S
VS
Department of Medicine, University of California, Irvine, California, USA.
Marchant
Jonathan S
JS
Ye
Dongmei
D
Ma
Thomas Y
TY
Said
Hamid M
HM
D000818:Animals; D018938:Caco-2 Cells; D002118:Calcium / Q000378:metabolism; D016764:Cell Polarity; D003078:Colchicine / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000187:drug effects; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D015739:Nocodazole / Q000494:pharmacology; D062793:Occludin; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013696:Temperature; D019108:Tight Junctions / Q000378:metabolism*; D014162:Transfection; D022161:Transport Vesicles / Q000378:metabolism; D050257:Tubulin Modulators / Q000494:pharmacology
D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D050257:Tubulin Modulators; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D015638:Cytochalasin D; D008274:Magnesium; D015739:Nocodazole; D003078:Colchicine; D002118:Calcium