Endocytosis of Epithelial Apical Junctional Proteins by a Clathrin-mediated Pathway into a Unique Storage Compartment

TLDR

Adherens and tight junctions maintain epithelial polarity and barrier function, and their loss is associated with endocytosis of these junctions, a process that may influence tissue remodeling and disease. The study sought to delineate the internalization pathway of AJ and TJ proteins in T84 epithelial cells using a calcium‑depletion model. Calcium depletion induced internalization of E‑cadherin, p120, β‑catenins, occludin, JAM‑1, claudins 1 and 4, and ZO‑1, and the authors tracked their trafficking through cellular compartments. Proteinase protection and immunocytochemistry showed that AJ and TJ proteins are endocytosed via clathrin‑mediated pathways into a subapical compartment that progresses to syntaxin‑4–positive organelles but not to late or recycling endosomes, lysosomes, or the Golgi, establishing a unique storage compartment.

Abstract

The adherens junction (AJ) and tight junction (TJ) are key regulators of epithelial polarity and barrier function. Loss of epithelial phenotype is accompanied by endocytosis of AJs and TJs via unknown mechanisms. Using a model of calcium depletion, we defined the pathway of internalization of AJ and TJ proteins (E-cadherin, p120 and beta-catenins, occludin, JAM-1, claudins 1 and 4, and ZO-1) in T84 epithelial cells. Proteinase protection assay and immunocytochemistry revealed orchestrated internalization of AJs and TJs into a subapical cytoplasmic compartment. Disruption of caveolae/lipid rafts did not prevent endocytosis, nor did caveolin-1 colocalize with internalized junctional proteins. Furthermore, AJ and TJ proteins did not colocalize with the macropinocytosis marker dextran. Inhibitors of clathrin-mediated endocytosis blocked internalization of AJs and TJs, and junctional proteins colocalized with clathrin and alpha-adaptin. AJ and TJ proteins were observed to enter early endosomes followed by movement to organelles that stained with syntaxin-4 but not with markers of late and recycling endosomes, lysosomes, or Golgi. These results indicate that endocytosis of junctional proteins is a clathrin-mediated process leading into a unique storage compartment. Such mechanisms may mediate the disruption of intercellular contacts during normal tissue remodeling and in pathology.