Protein interactions at the tight junction. Actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3.
Defining how the molecular constituents of the tight junction interact is a prerequisite to understanding tight junction physiology. We utilized in vitro binding assays with purified recombinant proteins and immunoprecipitation analyses to define interactions between ZO-1, ZO-2, ZO-3, occludin, and the actin cytoskeleton. Actin cosedimentation studies showed that ZO-2, ZO-3, and occludin all interact directly with F-actin in vitro, indicating that actin is engaged in multiple interactions at the tight junction. Low speed sedimentation analyses demonstrated that neither ZO-2, ZO-3, nor occludin act as F-actin cross-linking proteins, and further evidence indicates that these proteins do not bind to actin filament ends. The binding interactions of ZO-2, ZO-3, and occludin were corroborated in vivo by immunofluorescence colocalization experiments which showed that all three proteins colocalized with actin aggregates at cell borders in cytochalasin D-treated Madin-Darby canine kidney cells. Exploration of other tight junction protein interactions demonstrated that ZO-2 binds directly to both ZO-1 and occludin. Contrary to previous beliefs, our immunoprecipitation results indicate that ZO-1, ZO-2, and ZO-3 exist in situ primarily as independent ZO-1.ZO-2 and ZO-1.ZO-3 complexes rather than a trimeric ZO-1.ZO-2.ZO-3 grouping. These studies elucidate direct binding interactions among tight junction-associated proteins, giving insight into their organization as a multimolecular structure.
Wittchen
E S
ES
Department of Cell Biology, University of Alberta, Edmonton T6G 2H7, Canada.
Haskins
J
J
Stevenson
B R
BR
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002846:Chromatography, Affinity; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects; D004285:Dogs; D008565:Membrane Proteins / Q000378:metabolism*; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D019108:Tight Junctions / Q000378:metabolism*; D062825:Zonula Occludens Proteins; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein
D000199:Actins; D002352:Carrier Proteins; D008565:Membrane Proteins; D062793:Occludin; D010750:Phosphoproteins; D011994:Recombinant Proteins; D062825:Zonula Occludens Proteins; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; D015638:Cytochalasin D