Abstract
ZO-2, a member of the MAGUK family, was thought to be specific for tight junctions (TJs) in contrast to ZO-1, another MAGUK family member, which is localized at TJs and adherens junctions (AJs) in epithelial and nonepithelial cells, respectively. Mouse ZO-2 cDNA was isolated, and a specific polyclonal antibody was generated using corresponding synthetic peptides as antigens. Immunofluorescence microscopy with this polyclonal antibody revealed that, similarly to ZO-1, in addition to TJs in epithelial cells, ZO-2 was also concentrated at AJs in nonepithelial cells such as fibroblasts and cardiac muscle cells lacking TJs. When NH2-terminal dlg-like and COOH-terminal non-dlg-like domains of ZO-2 (N-ZO-2 and C-ZO-2, respectively) were separately introduced into cultured cells, N-ZO-2 was colocalized with endogenous ZO-1/ZO-2, i.e. at TJs in epithelial cells and at AJs in non-epithelial cells, whereas C-ZO-2 was distributed along actin filaments. Consistently, occludin as well as alpha catenin directly bound to N-ZO-2 as well as the NH2-terminal dlg-like portion of ZO-1 (N-ZO-1) in vitro. Furthermore, immunoprecipitation experiments revealed that the second PDZ domain of ZO-2 was directly associated with N-ZO-1. These findings indicated that ZO-2 forms a complex with ZO-1/occludin or ZO-1/alpha catenin to establish TJ or AJ domains, respectively.
Highlights
Generation and maintenance of specialized membrane domains are required for cells to exert their physiological functions, and the underlying molecular mechanisms of these processes are attracting increasing interest from cell biologists
NH2terminal dlg-like portion of ZO-1 (N-ZO-1) and N-ZO-2 were produced in Sf9 cells by baculovirus infection, and the cell lysate of Sf9 cells containing almost the same amounts of N-ZO-1 or N-ZO-2 was incubated with a GST fusion protein with the cytoplasmic domain of occludin (Fig. 5a)
When HA-tagged C-ZO-2 cDNA was introduced into NZ-EL cells or CZ-EL cells, neither N-ZO-1 or C-ZO-1 was co-immunoprecipitated with C-ZO-2, indicating that the NH2-terminal dlg-like domain of ZO-2 is responsible for ZO-1 binding
Summary
Generation and maintenance of specialized membrane domains are required for cells to exert their physiological functions, and the underlying molecular mechanisms of these processes are attracting increasing interest from cell biologists. ZO-1 was first identified as a peripheral membrane protein with a molecular mass of 220 kDa and was concentrated at TJs in epithelial cells [17, 18].
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