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Association between segments of zonula occludens proteins: live‐cell FRET and mass spectrometric analysis
Author(s) -
Rueckert Christine,
Castro Victor,
Gagell Corinna,
Dabrowski Sebastian,
Schümann Michael,
Krause Eberhard,
Blasig Ingolf E.,
Haseloff Reiner F.
Publication year - 2012
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1111/j.1749-6632.2012.06571.x
Subject(s) - tight junction , förster resonance energy transfer , microbiology and biotechnology , hek 293 cells , fusion protein , scaffold protein , chemistry , immunoprecipitation , green fluorescent protein , recombinant dna , transfection , cell junction , mutant , biophysics , cell , biology , fluorescence , biochemistry , signal transduction , gene , physics , quantum mechanics
The tight junction protein ZO‐1 (zonula occludens protein 1) has recruiting/scaffolding functions in the junctional complex of epithelial and endothelial cells. Homodimerization was proposed to be crucial for ZO‐1 function. Here, we investigated the ability of ZO‐1 domains to mediate self‐interaction in living cells. We expressed ZO‐1 truncation mutants as fusions with derivatives of green fluorescent protein in tight junction–free HEK‐293 cells and determined self‐association by means of fluorescence resonance energy transfer measurements using live‐cell imaging. We show that both an SH3‐hinge‐GuK fusion protein and the PDZ2 domain self‐associate in our test system. The recombinant PDZ2 domain also binds to ZO‐1 and ZO‐2 in tight junction–forming HT29/B6 cell lysates, as demonstrated by coprecipitation. Both interaction types are of relevance for the function of ZO‐1 in the regulation of the junctional complex in polar cells.