Tight junctions are composed of multimeric protein complexes often formed by different types of proteins that interact with each other. Epithelial junction formation is a sequential process that starts with the formation of a primordial adhesive junction that then matures into distinct tight and adherens junctions. ZO-1 can interact with different components of tight and adherens junctions and appears to play a modulatory role during formation of both types of junctions [16, 17, 36]. ZO-1 also regulates gene expression, cell proliferation and epithelial morphogenesis [18–20]. ZO-1's role in gene expression is regulated by an interaction with the heat shock protein Apg-2 . Here we show that Apg-2 also modulates junction formation and is required for normal epithelial morphogenesis in 3-D cultures.
Depletion of Apg-2 affects junction formation in a similar way as depletion of ZO-1: formation of functional junctions was retarded, but not prevented, and the inhibition was only modest. In the case of ZO-1, it seems that its function in tight junction formation is at least in part redundant as depletion of ZO-2 in ZO-1 knockout cells prevents tight junction formation . Whether MDCK cells express other proteins with a similar function as Apg-2 is currently not known; however, it is conceivable that other heat shock proteins might also aid junction formation. Alternatively, it could be that Apg-2 functions as a catalyst; hence, its function is not required, but its presence accelerates junction formation.
The mechanism by which Apg-2 regulates junction assembly is not known. It could be that Apg-2 regulates formation of ZO-1 complexes. We have thus far not been able, however, to detect differences in proteins co-precipitating with ZO-1 if Apg-2 was depleted (KM and MSB, unpublished). Nevertheless, it is possible that this involves interactions that are not detergent resistant or other unknown proteins. We could also not detect clear differences in the Triton X-100 insolubility of junctional proteins between control and Apg-2 depleted cells, but there was only a small pool of insoluble proteins at early time points when junction formation was inhibited (not shown). As we have only observed kinetic differences in junction formation, minor quantitative changes in complex formation might be involved that would be difficult to assess experimentally.
The SH3 domain of ZO-1, which is the domain that interacts with Apg-2, is not only required but also sufficient to rescue junction formation in ZO-1 depleted MDCK cells [16, 21]. However, the SH3 domain by itself remains in the cytosol and is not recruited to cell junctions [18, 19], suggesting that it binds to a cellular factor that does not need to be recruited to junctions. One such factor is the transcription factor ZONAB; however, neither overexpression nor depletion of ZONAB affects tight junction formation (KM and MSB, unpublished) . Hence, one possibility is that ZO-1 regulates a function of Apg-2 rather than the other way around. In fact, ZO-1 does not bind the peptide-binding domain of Apg-2 but to the N-terminal domain containing the ATPase. Hence, ZO-1 binding might affect yet to be discovered functional properties of Apg-2 such as, for example, release of another protein bound to the peptide-binding domain. It will therefore be important to analyse how ZO-1 binding affects the biochemical properties of Apg-2 and to search for Apg-2 substrates and other interacting proteins.
Apg-2 also regulates epithelial morphogenesis in 3-D cultures. Apg-2 depleted cells did not form hollow cysts but poorly organised structures with none or several small lumen. The disorganised nature of these structures was evident from different immunofluorescence labellings that showed that their cells did not possess a uniform polarity or, in the absence of a clear lumen, maintained apical markers at the outer surface of the cysts. Strikingly, we observed the same phenotype in cells depleted of ZO-1. As overexpression of ZONAB also caused a similar phenotype, we considered it as likely that the reduced expression of ZO-1 affected morphogenesis by stimulating ZONAB . Apg-2 depletion, however, does not stimulate ZONAB but inhibits it, as the two proteins compete with each other for binding to the SH3 domain of ZO-1 . However, it is possible that deregulation, and not just activation, of the ZO-1/ZONAB pathway causes defects in the development of polarised hollow cysts. Alternatively, ZO-1 might regulate a function of Apg-2 that is important for morphogenesis. Moreover, Apg-2 is expressed in different subcellular locations and, like other heat shock proteins, might bind many different proteins and, hence, regulate different types of processes. Nevertheless, the similarity of the phenotypes in junction formation and 3-D morphogenesis caused by depletion of Apg-2 and ZO-1 suggests that the two interacting proteins function in at least overlapping pathways during epithelial differentiation.