The Tight Junction Protein ZO-1 Establishes a Link between the Transmembrane Protein Occludin and the Actin Cytoskeleton

TLDR

ZO‑1 is a membrane‑associated guanylate kinase homolog that interacts with occludin, ZO‑2, and F‑actin, yet the functional significance of these interactions remains poorly understood. The study aims to delineate ZO‑1’s role in epithelial tight junctions by expressing epitope‑tagged ZO‑1 fragments in MDCK cells to identify domains mediating interactions with ZO‑2, occludin, and F‑actin. Using in vitro and in vivo binding assays on epitope‑tagged ZO‑1 fragments expressed in MDCK cells, the authors mapped interaction domains with ZO‑2, occludin, and F‑actin. The N‑terminal MAGUK‑like half of ZO‑1 binds ZO‑2 and occludin, the C‑terminal proline‑rich half binds F‑actin and localizes to the lateral membrane, a 244‑aa N‑terminal domain is required for stable junctional incorporation, and together these interactions enable ZO‑1 to organize tight junction components and link them to the cortical actin cytoskeleton.

Abstract

The tight junction protein ZO-1 belongs to a family of multidomain proteins known as the membrane-associated guanylate kinase homologs (MAGUKs). ZO-1 has been demonstrated to interact with the transmembrane protein occludin, a second tight junction-specific MAGUK, ZO-2, and F-actin, although the nature and functional significance of these interactions is poorly understood. To further elucidate the role of ZO-1 within the epithelial tight junction, we have introduced epitope-tagged fragments of ZO-1 into cultured MDCK cells and identified domains critical for the interaction with ZO-2, occludin, and F-actin. A combination of in vitro and in vivo binding assays indicate that both ZO-2 and occludin interact with specific domains within the N-terminal (MAGUK-like) half of ZO-1, whereas the unique proline-rich C-terminal half of ZO-1 cosediments with F-actin. Consistent with these observations, we found that a construct encoding the N-terminal half of ZO-1 is specifically associated with tight junctions, whereas the unique C-terminal half of ZO-1 is distributed over the entire lateral surface of the plasma membrane and other actin-rich structures. In addition, we have identified a 244-amino acid domain within the N-terminal half of ZO-1, which is required for the stable incorporation of ZO-1 into the junctional complex of polarized MDCK cells. These observations suggest that one functional role of ZO-1 is to organize components of the tight junction and link them to the cortical actin cytoskeleton.