title: Identification of adherens junction-associated GTPase activating proteins by the fluorescence localization-based expression cloning.
abstract: The junctional complex, including tight junctions (TJs), adherens junctions (AJs), and desmosomes, plays crucial roles in the structure and functions of epithelial cellular sheets. In this study, we evaluated the fluorescence localization-based retrovirus-mediated expression cloning (FL-REX) method as an approach to identify novel molecular components of TJs and AJs. Using an expression library of cDNA-GFP-fusions derived from mRNA of a mouse epithelial cell line, we confirmed that cDNAs for various known TJ- and AJ-components could be cloned in the FL-REX. Furthermore, cDNAs for ARHGAP12 and SPAL3, two putative GTPase activating proteins (GAPs) for small G proteins, were cloned as novel components of the junctional complex. Immunofluorescence staining using antibodies generated in-house demonstrated that these GAPs were localized at epithelial cell-cell junctions in various mouse tissues, and were specific to AJs when observed under confocal laser-scanning microscopy. These data suggest that FL-REX is a powerful tool to identify novel proteins localized at TJs and AJs.
authors.affiliation: Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Miho
authors.initials: M
authors.lastname: Matsuda
authors.forename: Yuka
authors.initials: Y
authors.lastname: Kobayashi
authors.forename: Sayuri
authors.initials: S
authors.lastname: Masuda
authors.forename: Makoto
authors.initials: M
authors.lastname: Adachi
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Watanabe
authors.forename: Jun K
authors.initials: JK
authors.lastname: Yamashita
authors.forename: Eiichiro
authors.initials: E
authors.lastname: Nishi
authors.forename: Shoichiro
authors.initials: S
authors.lastname: Tsukita
authors.forename: Mikio
authors.initials: M
authors.lastname: Furuse
mesh_terms: D022005:Adherens Junctions / Q000737:chemistry*; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004847:Epithelial Cells / Q000648:ultrastructure; D005453:Fluorescence; D020690:GTPase-Activating Proteins / Q000032:analysis*; D051379:Mice; D019108:Tight Junctions / Q000737:chemistry
chemical_list: D020690:GTPase-Activating Proteins