Identification of adherens junction-associated GTPase activating proteins by the fluorescence localization-based expression cloning.
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.
Matsuda
Miho
M
Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Kobayashi
Yuka
Y
Masuda
Sayuri
S
Adachi
Makoto
M
Watanabe
Tsuyoshi
T
Yamashita
Jun K
JK
Nishi
Eiichiro
E
Tsukita
Shoichiro
S
Furuse
Mikio
M
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
D020690:GTPase-Activating Proteins