Abstract
Claudins (Cldns) are transmembrane tight junction (TJ) proteins that paracellularly seal endo- and epithelial barriers by their interactions within the TJs. However, the mechanisms allowing TJ remodeling while maintaining barrier integrity are largely unknown. Cldns and occludin are heterophilically and homophilically cross-over endocytosed into neighboring cells in large, double membrane vesicles. Super-resolution microscopy confirmed the presence of Cldns in these vesicles and revealed a distinct separation of Cldns derived from opposing cells within cross-over endocytosed vesicles. Colocalization of cross-over endocytosed Cldn with the autophagosome markers as well as inhibition of autophagosome biogenesis verified involvement of the autophagosomal pathway. Accordingly, cross-over endocytosed Cldns underwent lysosomal degradation as indicated by lysosome markers. Cross-over endocytosis of Cldn5 depended on clathrin and caveolin pathways but not on dynamin. Cross-over endocytosis also depended on Cldn-Cldn-interactions. Amino acid substitutions in the second extracellular loop of Cldn5 (F147A, Q156E) caused impaired cis- and trans-interaction, as well as diminished cross-over endocytosis. Moreover, F147A exhibited an increased mobility in the membrane, while Q156E was not as mobile but enhanced the paracellular permeability. In conclusion, the endocytosis of TJ proteins depends on their ability to interact strongly with each other in cis and trans, and the mobility of Cldns in the membrane is not necessarily an indicator of barrier permeability. TJ-remodeling via cross-over endocytosis represents a general mechanism for the degradation of transmembrane proteins in cell-cell contacts and directly links junctional membrane turnover to autophagy.
Highlights
Tight junctions (TJs) are mainly formed by claudins (Cldns), transmembrane proteins that play a major role in limiting and regulating paracellular permeation in epithelia and endothelia
A direct involvement of the autophagic machinery in TJ-remodeling is demonstrated, and for the first time the presence of Cldns is shown in large double membrane vesicles
Our results confirm a type of endocytosis of Cldns, where TJ-associated Cldn-oligomers are cross-over endocytosed into the neighboring cell in double-membrane vesicles in a clathrin- and caveolin-dependent manner
Summary
Tight junctions (TJs) are mainly formed by claudins (Cldns), transmembrane proteins that play a major role in limiting and regulating paracellular permeation in epithelia and endothelia. Calcium depletion in epithelial T84 cells leads to clathrin-dependent internalization of Cldns 1 and 4 into subapical ring-like organelles that are positive for syntaxin-4, but not for markers of lysosomes, the Golgi apparatus, or late or recycling endosomes [10]. These data indicate that there may be a poorly characterized compartment involved in Cldn internalization. While there is an abundance of information regarding stimuli leading to TJ disruption, there is only limited knowledge on constitutive internalization pathways of TJ proteins
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