ZO-1 knockout by TALEN-mediated gene targeting in MDCK cells: involvement of ZO-1 in the regulation of cytoskeleton and cell shape.

Shinsaku Tokuda,Tomohito Higashi,Mikio Furuse,Mirjam M Zegers

doi:10.1371/journal.pone.0104994

Shinsaku Tokuda, Tomohito Higashi + Show 2 more

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https://doi.org/10.1371/journal.pone.0104994

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Abstract

ZO-1, ZO-2 and ZO-3 are tight junction-associated scaffold proteins that bind to transmembrane proteins of tight junctions and the underlying cytoskeleton. ZO-1 is involved in the regulation of cytoskeletal organization, but its detailed molecular mechanism is less well understood. Gene knockout is an ideal method to investigate the functions of proteins that might have redundant functions such as ZO proteins, when compared with methods such as RNA interference-mediated suppression of gene expression. In this study we applied transcription activator-like effector nucleases (TALENs), a recently developed genome editing method for gene knockout, and established ZO-1 knockout clones in Madin-Darby canine kidney (MDCK) cells. ZO-1 knockout induced striking changes in myosin organization at cell–cell contacts and disrupted the localization of tight junction proteins; these findings were previously unseen in studies of ZO-1 knockdown by RNA interference. Rescue experiments revealed that trace ZO-1 expression reversed these changes while excessive ZO-1 expression induced an intensive zigzag shape of cell–cell junctions. These results suggest a role for ZO-1 in the regulation of cytoskeleton and shape of cell–cell junctions in MDCK cells and indicate the advantage of knockout analysis in cultured cells.

Highlights

IntroductionEpithelia act as a barrier to the external environment. Epithelial cells adhere to each other through complexes that form junctions between the cells, and the tight junction (TJ) is located in the most apical part of the complexes [1]
In multicellular organisms, epithelia act as a barrier to the external environment
Staining of ZO-2 at tight junction (TJ) was not altered in zonula occludens-1 (ZO-1) knockout Madin-Darby canine kidney (MDCK) I cells but ZO-3 staining at TJs was markedly reduced in ZO-1 knockout MDCK I cells compared with control cells (Fig. 2C and D)

Summary

Introduction

Epithelia act as a barrier to the external environment. Epithelial cells adhere to each other through complexes that form junctions between the cells, and the tight junction (TJ) is located in the most apical part of the complexes [1]. TJs regulate movements of several substances through the paracellular pathway to maintain homeostasis in the internal environment required for proper organ function [2,3]. ZO proteins are likely to be important for the regulation of the cytoskeleton at TJs, and consistent with this idea, the suppression of ZO-1 expression by RNA interference (knockdown) in Madin-Darby canine kidney (MDCK) II cells was reported to change the organization of the perijunctional cytoskeleton and the shape of cell–cell junctions from tortuous to linear [13]. Two other studies did not note these changes in ZO-1 knockdown MDCK II cells [14,15]. Because RNAimediated knockdown is not complete and only reduces gene function, one explanation for the differences observed between these studies might be differences in the levels of remaining ZO-1 expression [13]

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