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Real time analysis of TNF‐induced occludin internalization within jejunal epithelia of living mice
Author(s) -
Marchiando Amanda M,
Shen Le,
Guan Yanfang,
Watson Alastair J.M.,
Montrose Marshall H.,
Turner Jerrold R.
Publication year - 2007
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.21.5.a585-c
Subject(s) - occludin , internalization , microbiology and biotechnology , tight junction , endocytosis , biology , transcytosis , caveolae , dynamin , in vivo , chemistry , biochemistry , signal transduction , cell
In vitro and in vivo studies have shown that TNF causes intestinal epithelial barrier dysfunction. This effect is cytoskeletally‐mediated and associated with redistribution of the tight junction (TJ) protein occludin to intracellular endocytic compartments. However, technical limitations have prevented real‐time analyses of occludin internalization. Our aim was to use in vivo imaging to characterize the process of TNF‐induced occludin internalization. We developed transgenic mice expressing an EGFP‐occludin fusion protein driven by the villin promoter. EGFP‐occludin colocalized with endogenous occludin and expression was restricted to the intestinal epithelium. Jejunal mucosa of anesthetized mice was surgically exposed and imaged by confocal microscopy. EGFP‐occludin internalization began 90 min after intraperitoneal TNF injection (5μg). The first observed change was focal increases in occludin concentration within the TJ. Small vesicles then invaginated from these occludin‐rich zones, budding from the basal aspect of the TJ. Immunofluorescent colocalization studies showed that 31% of EGFP‐occludin‐containing vesicles were positive for caveolin‐1, while only 6% were positive for clathrin heavy chain. These data are the first to image the process of TJ protein internalization. Moreover, they suggest that TNF‐induced occludin endocytosis occurs via a caveolae‐mediated pathway in vivo . Supported by NIDDK.