Establishment and characterization of cultured epithelial cells lacking expression of ZO-1.
In well polarized epithelial cells, closely related ZO-1 and ZO-2 are thought to function as scaffold proteins at tight junctions (TJs). In epithelial cells at the initial phase of polarization, these proteins are recruited to cadherin-based spotlike adherens junctions (AJs). As a first step to clarify the function of ZO-1, we successfully generated mouse epithelial cell clones lacking ZO-1 expression (ZO-1-/- cells) by homologous recombination. Unexpectedly, in confluent cultures, ZO-1-/- cells were highly polarized with well organized AJs/TJs, which were indistinguishable from those in ZO-1+/+ cells by electron microscopy. In good agreement, by immunofluorescence microscopy, most TJ proteins including claudins and occludin appeared to be normally concentrated at TJs of ZO-1-/- cells with the exception that a ZO-1 deficiency significantly up- or down-regulated the recruitment of ZO-2 and cingulin, another TJ scaffold protein, respectively, to TJs. When the polarization of ZO-1-/- cells was initiated by a Ca2+ switch, the initial AJ formation did not appear to be affected; however, the subsequent TJ formation (recruitment of claudins/occludin to junctions and barrier establishment) was markedly retarded. This retardation as well as the disappearance of cingulin were rescued completely by exogenous ZO-1 but not by ZO-2 expression. Quantitative evaluation of ZO-1/ZO-2 expression levels led to the conclusion that ZO-1 and ZO-2 would function redundantly to some extent in junction formation/epithelial polarization but that they are not functionally identical. Finally, we discussed advantageous aspects of the gene knock-out system with cultured epithelial cells in epithelial cell biology.
Umeda
Kazuaki
K
Department of Cell Biology, Kyoto University Faculty of Medicine, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan.
Matsui
Takeshi
T
Nakayama
Mayumi
M
Furuse
Kyoko
K
Sasaki
Hiroyuki
H
Furuse
Mikio
M
Tsukita
Shoichiro
S
D022005:Adherens Junctions; D000483:Alleles; D000818:Animals; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation; D018920:Coculture Techniques; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D004847:Epithelial Cells / Q000378:metabolism*; D005822:Genetic Vectors; D015151:Immunoblotting; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D062793:Occludin; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D011995:Recombination, Genetic; D013997:Time Factors; D019076:Transgenes; D015854:Up-Regulation; D062826:Zonula Occludens-1 Protein
C489227:Cgn protein, mouse; D018076:DNA, Complementary; D008565:Membrane Proteins; D062793:Occludin; C568986:Ocln protein, mouse; D010750:Phosphoproteins; C569043:Tjp1 protein, mouse; D062826:Zonula Occludens-1 Protein; D002118:Calcium