Date of Award

5-2021

Document Type

Thesis campus only

Department

Biology

First Advisor

Jonathan M. King

Second Advisor

Bethany Strunk

Third Advisor

Kwan K. Cheng

Abstract

The goal of this study was to create, isolate, and analyze a cell line of epithelial cells lacking the tight junction protein, ZO-1. The central hypothesis was that ZO-1 plays notable roles in permeability and proliferation and ZO-1 interaction with the actin cytoskeleton has a role in the regulation of cell membrane tension. The study developed ZO-1 null MDCK cell lines using CRISPR/Cas9 ribonucleoprotein complex. Additionally, the study examined phenotypic differences in wildtype (WT) and knockout (KO) cell lines by measuring protein expression as well as protein localization. Barrier properties were analyzed in WT and KO cell lines. Nuclear density in ZO-1 ablated cells was compared to WT using immunofluorescent staining and ImageJ macros. To study the relationship between altered cytoskeleton and loss of ZO-1 the study measured membrane tension using a fluorescent probe and FLIM analysis to evaluate mechanical properties in WT and KO cells. Cells lacking ZO-1 appear to be more nuclear dense, accumulate apical actin, have an increased cell height, and exhibit a higher membrane tension than their wild type counterparts. Notably, the increased membrane tension cause has not yet been determined but could have relation to the increased cell density or the increased apical actin noted in our knockout studies. The creation and isolation of a ZO-1 knockout line provides a basis for further studies into the specific functions of various domains and residues of ZO-1.

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