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Zonula occludens--‐1 (ZO--‐1) is a large scaffold protein necessary for tight junction formation and signaling. Despite extensive studies of the ZO--‐1 protein, there remains ambiguity concerning the function of the C--‐terminal ZU5 domain. In order to study the structure and function of the ZU5 domain, we subcloned a C--‐terminal, 333 amino acid sequence containing the ZU5 domain from mouse ZO--‐1 into the phrGFP II--‐N mammalian expression vector. This vector was subsequently used to create a stable line of MDCK II cells transfected with a phrGFP--‐ZU5 fusion construct. We found that the product of the ZU5 expression construct appropriately localizes to cell--‐cell boundaries. ZU5 expression alters physiological parameters including tight junction gate function and remodeling, as is evident through measurements of transepithelial electrical resistance (TER) and paracellular flux. The calcium switch assay demonstrated alteration in junctional formation when compared to the parent MDCK II cell line. While expression levels in these cells are high enough to allow for the visualization of ZU5 localization using fluorescence microscopy, they are too low for biochemical analysis. In order to pursue such studies, we developed various C--‐terminal constructs (ABR, ABR--‐PRO, PRO--‐ZU5, and ZU5) from mouse ZO--‐1 and created stable lines of MDCK II cells transfected with 3xFlag--‐tagged proteins. Expression in these cell lines resulted in altered tight junction gate function, remodeling and assembly following calcium depletion. Biochemical analyses and binding assays have not yet been completed. However, from our preliminary functional data, we suggest an intramolecular binding mechanism in the C--‐terminal region of ZO--‐1. The development of stable cell lines in the present study will be fundamental for the continued study of the ZU5 domain and the rest of the c--‐terminal ZO--‐1 protein.
Brady, Rachel C., "Structure-‐function analysis of the C-‐terminal domain, ZU5, of the Zonula Occludens-‐1 protein" (2011). Biology Honors Theses. 7.
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