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Analysis of ZO‐1, ARHGEF11 and Actin Cytoskeleton at the Apical Junctional Complex
Author(s) -
White Addison R.,
Mudekunye Gabriella G.,
King Jonathan M.
Publication year - 2018
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.2018.32.1_supplement.624.36
Subject(s) - microbiology and biotechnology , actin cytoskeleton , cytoskeleton , biology , tight junction , actin , cell , biochemistry
Background Zonula occludens (ZO‐1) is a tight junction cytosolic scaffolding protein known to bind the actin cytoskeleton in epithelial cells. ARHGEF11 is a Rho guanine nucleotide exchange factor known to activate the Rho family of small GTPases which are regulators of the actin cytoskeleton of the cell. ARHGEF11 associates with ZO‐1 by binding to the ZU5 domain in the C‐terminal tail. We are interested in understanding the interplay between ZO‐1, ARHGEF11 and the actin cytoskeleton in confluent MDCK II cells. Methods Confluent monolayers stably transfected with EGFP‐ZO‐1 or EGFP‐ZO‐1Δ ZU5 were treated with latrunculin A to observe the actin depolymerization protection capacity of ZO‐1 with and without the ZU5 domain. Additionally, studies were performed to investigate the localization of EGFP‐ZO‐1 or EGFP‐ZO‐1ΔZU5 proteins following recovery from latrunculin A exposure. Colocalization studies were performed in mCherry‐ARHGEF11/EGFP‐ZO‐1 co‐transfected cells. Pharmacological inhibition of Rho‐kinase was used to target the ARHGEF11 pathway. Electrophysiological studies were conducted to evaluate junctional permeability. Results Full‐length EGFP‐ZO‐1 protects against latrunculin A induced actin depolymerization more robustly than EGFP‐ZO‐1ΔZU5. In the actin polymerization recovery paradigm EGFP‐ZO‐1ΔZU5 exhibited enhanced recovery rate when compared to full‐length EGFP‐ZO‐1 following latrunculin A exposure. Enhanced actin networks are observed in mCherry‐ARHGEF11 positive cells in both wildtype and ZO‐1 knockdown MDCK II cells. Conclusions The increased recovery rate exhibited by EGFP‐ZO‐1ΔZU5 in the latrunculin A recovery assay coupled with the greater actin protection capacity of full‐length ZO‐1 suggests that truncated ZO‐1 is more mobile. ARHGEF11 elicits actin cytoskeleton reorganization in both wildtype and ZO‐1 knockdown cells that was partially reversed by inhibition of Rho kinase. There was limited accumulation of ARHGEF11 at the apical junctional complex in confluent monolayers. Support or Funding Information San Antonio Medical Foundation, Trinity University Biology Department, Mach Research Award This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .