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Physiological Analysis of the ZO‐1 Actin Binding Region on Cell Adhesion and Function in Epithelial Cells
Author(s) -
Loar Abigail M,
Winkler Garry A,
Brady Rachel C,
King Jonathan M
Publication year - 2012
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.26.1_supplement.905.8
Subject(s) - microbiology and biotechnology , transfection , fusion protein , barrier function , cell adhesion , tight junction , green fluorescent protein , chemistry , actin , cell , biology , cell culture , recombinant dna , biochemistry , genetics , gene
Zonula occludens‐1 (ZO‐1) is a large junctional scaffold protein within epithelium that contributes to the regulation between a proliferative and differentiated state. The function of the ZO‐1 Actin Binding Region (ABR) was examined by overexpression of either GFP‐ABR‐ZO‐1 or 3XFlag‐ABR‐ZO‐1 fusion constructs into stably transfected MDCK II cells. Additionally, 6XHis‐tagged‐ ABR‐ZO‐1 was used to produce recombinant ABR protein to examine molecular interactions with the ABR domain. Calcium switch experiments demonstrated the 3XFlag‐ABR‐ZO‐1 overexpression significantly enhanced barrier function and kinetics of tight junction assembly after an initial lag phase. In live cell imaging experiments, GFP‐ABR‐ZO‐1 markedly altered MDCK cell phenotype into spindle‐like cells deviating from the expected epithelial monolayer and adhesive properties. To study multicellular aggregation in a physiologically relevant system, MDCK cells were induced to form cysts by three‐dimensional cell culture in Geltrex extracellular matrix. GFP‐ABR‐ZO‐1 overexpression showed unusual morphogenesis, with unorganized cysts more than twice the size of control MDCK cysts. These studies highlight the ZO‐1‐ABR region as an important regulator of cell morphology, adhesion, and multicellular epithelial organization.