ZO-1 mRNA and protein expression during tight junction assembly in Caco-2 cells.
We previously identified and characterized ZO-1 as a peripheral membrane protein specifically associated with the cytoplasmic surface of tight junctions. Here we describe the identification of partial cDNA sequences encoding rat and human ZO-1 and their use to study the assembly of tight junctions in the Caco-2 human intestinal epithelial cell line. A rat cDNA was isolated from a lambda-gtll expression library by screening with mAbs. Polyclonal antibodies were raised to cDNA-encoded fusion protein; several properties of these antibodies support this cDNA as encoding ZO-1. Expression of ZO-1 mRNA occurs in the rat and Caco-2 cells with a major transcript of approximately 7.5 kb. To disrupt tight junctions and study the subsequent process of assembly, Caco-2 cells were grown in suspension for 48 h in Ca++/Mg++-free spinner medium during which time they lose cell-cell contacts, become round, and by immunofluorescence microscopy show diffuse and speckled localization of ZO-1. Within hours of replating at confluent density in Ca++/Mg++-containing media, attached cells show discrete localization of ZO-1 at cell-cell contacts. Within 2 d, fully confluent monolayers form, and ZO-1 localizes in a continuous gasket-like fashion circumscribing all cells. ZO-1 mRNA levels are highest in cells in spinner culture, and upon replating rapidly fall and plateau at approximately 10% of initial levels after 2-3 wk in culture. ZO-1 protein levels are lowest in contact-free cells and rise five- to eightfold over the same period. In contrast, mRNA levels for sucrase-isomaltase, an apical membrane hydrolase, increase only after a confluent monolayer forms. Thus, in this model of contact-dependent assembly of the tight junction, there is both a rapid assembly beginning upon cell-cell contact, as well as a long-term modulation involving changes in expression of ZO-1 mRNA and protein levels.
Anderson
J M
JM
Department of Internal Medicine, Yale School of Medicine, New Haven, Connecticut 06510.
Van Itallie
C M
CM
Peterson
M D
MD
Stevenson
B R
BR
Carew
E A
EA
Mooseker
M S
MS
D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D046508:Culture Techniques / Q000379:methods; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D007365:Intercellular Junctions / Q000502:physiology* / Q000648:ultrastructure; D008099:Liver / Q000378:metabolism / Q000648:ultrastructure; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D010750:Phosphoproteins / Q000032:analysis / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D014158:Transcription, Genetic; D062826:Zonula Occludens-1 Protein
D008565:Membrane Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; C569042:TJP1 protein, human; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; D004247:DNA