A ZO1-GFP fusion protein to study the dynamics of tight junctions in living cells.
To examine the dynamics of tight junctions (TJs) in living cells, chimera between the TJ-associated protein ZO-1 and green fluorescent protein (GFP) were constructed. If ZO-1 fused to the C-terminus of GFP (ZO1-CGFP) was stably expressed in MDCK cells, it was fully incorporated into TJs and colocalized with endogenous ZO-1. The GFP tag did not influence cell growth, transepithelial electrical resistance, and paracellular mannitol transport. The morphology of the transfected cells was unchanged. The ZO1-CGFP MDCK cell line thus represents an excellent tool to study TJ dynamics. The influence of the external calcium ion concentration on the formation and dynamics of TJs in living cells was thus explored. Upon opening of the TJs under short-term treatment with EGTA (up to 20 min), the localization of ZO1-CGFP at the membrane persisted. The rim-like pattern around the individual cells appeared fuzzier than in non-treated cells. Long-term calcium depletion resulted in the localization of ZO1-CGFP in the cytoplasm and the nucleus. After restoration of normal Ca(2+) concentrations, cell-cell contacts were restored and the localization of ZO1-CGFP was indistinguishable from the one in control cells kept at normal Ca(2+) concentrations. It remains open how the different localizations of ZO-1 correspond to changes in the signal transduction activity of the molecule.
Riesen
Franziska K
FK
Biopharmacy, Department of Applied BioSciences, ETH Zurich, 8057 Zurich, Switzerland.
Rothen-Rutishauser
Barbara
B
Wunderli-Allenspach
Heidi
H
D000818:Animals; D002118:Calcium / Q000172:deficiency / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004285:Dogs; D017097:Electric Impedance; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism*; D007668:Kidney; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008353:Mannitol / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D019108:Tight Junctions / Q000378:metabolism*; D014162:Transfection; D062826:Zonula Occludens-1 Protein
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D062826:Zonula Occludens-1 Protein; D049452:Green Fluorescent Proteins; D008353:Mannitol; D002118:Calcium