title: ZO-1 knockout by TALEN-mediated gene targeting in MDCK cells: involvement of ZO-1 in the regulation of cytoskeleton and cell shape.
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.
authors.affiliation: Division of Cell Biology, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Tokuda
authors.affiliation: Division of Cell Biology, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Tomohito
authors.initials: T
authors.lastname: Higashi
authors.affiliation: Division of Cell Biology, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe, Japan; Division of Cerebral Structure, National Institute for Physiological Sciences, Okazaki, Japan.
authors.forename: Mikio
authors.initials: M
authors.lastname: Furuse
mesh_terms: D000818:Animals; D001483:Base Sequence; D048430:Cell Shape; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D004285:Dogs; D055785:Gene Knockdown Techniques / Q000379:methods*; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D061985:Madin Darby Canine Kidney Cells / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D008969:Molecular Sequence Data; D019108:Tight Junctions / Q000378:metabolism / Q000648:ultrastructure; D062826:Zonula Occludens-1 Protein / Q000235:genetics* / Q000378:metabolism; D062827:Zonula Occludens-2 Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein