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Caveolar endocytosis is essential for tumor necrosis factor (TNF) ‐induced occludin internalization in vivo
Author(s) -
Marchiando Amanda M,
Shen Le,
Schwarz Brad T.,
Guan Yanfang,
Watson Alastair JM,
Montrose Marshall H.,
Turner Jerrold R.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.938.5
Subject(s) - occludin , endocytosis , microbiology and biotechnology , internalization , dynamin , transcytosis , clathrin , tight junction , endocytic cycle , bulk endocytosis , caveolae , biology , chemistry , biochemistry , signal transduction , cell
TNF causes epithelial barrier dysfunction in vitro and in vivo and is accompanied by redistribution of the tight junction protein occludin. Mechanistic characterization of this vesicular trafficking process and associated tight junction regulation has been limited by the inability to image occludin internalization in real time. Our aim was to characterize the route and mechanisms of TNF‐induced occludin endocytosis in vivo . We developed mice expressing EGFP‐occludin and mRFP1‐ZO‐1 in intestinal epithelium, and both fusion proteins colocalized with their endogenous counterparts. Jejunal mucosa was imaged by confocal microscopy of live anaesthetized mice. TNF (5μg i.p.) triggered focal intra‐junctional occludin aggregation followed by occludin endocytosis. The small endocytic vesicles contained caveolin‐1 but not clathrin heavy chain. Occludin endocytosis was prevented by dynasore, a dynamin II inhibitor; L‐ t ‐LacCer, a synthetic lipid inhibitor of caveolar endocytosis; and cholesterol chelation with MβCD; while neither macropinocytosis nor clathrin‐mediated endocytosis inhibitors blocked internalization. Occludin endocytosis was also defective in caveolin‐1 knockout mice, though TNF activated myosin normally. These data are the first to image occludin protein trafficking in vivo and demonstrate the pivotal role of caveolin‐1‐dependent caveolar endocytosis in this process. Supported by NIH.