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Cytosolic localization of YFP‐Ezrin‐CFP prevents participation in the acid secretory pathway
Author(s) -
Delaney Joseph,
Zhu Lixin,
Forte John G.
Publication year - 2006
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.20.5.lb21-b
Subject(s) - ezrin , radixin , moesin , microbiology and biotechnology , chemistry , cytoplasm , secretion , cytoskeleton , biology , biochemistry , cell
The membrane cytoskeletal linker ezrin, a member of the Ezrin/Radixin/Moesin (ERM) family, has been hypothesized to have an inhibitory interaction between its N‐terminal FERM domain and C‐terminal ERM association domain (C‐ERMAD). The inactive state of ezrin has always been considered to have this head‐to‐tail association. Activation by phosphorylation was considered to relieve this intra‐ or intermolecular interaction. Ezrin is also required in gastric parietal cells to both stimulate acid secretion and form extend apical microvilli. In this study, fluorescence resonance energy transfer (FRET) compatible YFP‐Ezrin‐CFP (Y‐Ez‐C) was used to probe for the FERM to C‐ERMAD interaction. Parietal cells were infected with adenovirus containing this Y‐Ez‐C construct and then either held in a resting state with cimetidine or stimulated by adding histamine and IBMX. A weak FRET signal at 527 nm (425 nm ex.) was observed and identical for both resting and stimulated states. Unlike endogenous ezrin and cells expressing Ezrin‐CFP, which were primarily localized to the apical plasma membrane with some basolateral expression, Y‐Ez‐C was expressed throughout the cytoplasm. Aminopyrine assays revealed that Y‐Ez‐C infected cells stimulated acid secretion normally (97.8±9.2% of uninfected control), and confocal microscopy showed these cells to have normal time dependent apical vacuole swelling. These data indicate that the opening of the Y‐Ez‐C construct does not occur in vivo, whether or not secretion has been turned on, nor does it participate in the functions of endogenous ezrin to support parietal cell secretion.