Interplay between Anakonda, Gliotactin and M6 for tricellular junction assembly and anchoring of septate junctions in Drosophila epithelium

In epithelia, Tricellular junctions (TCJs) serve as pivotal sites for barrier function and integration of both biochemical and mechanical signals. While essential for tissue homeostasis, TCJ assembly, composition and links to adjacent bicellular junctions (BCJs) remain poorly understood. Here we have characterized the assembly of TCJs within the plane of adherens junctions (tAJ) and the plane of septate junctions (tSJ) in Drosophila and report that their formation is spatiotemporally decoupled. The assembly and stabilization of previously described tSJ components Anakonda (Aka) and Gliotactin (Gli) as well as the newly reported tSJ proteolipid protein M6, is shown to be a complex process. Aka and M6, whose localization is interdependent, act upstream to locate Gli. In turn, Gli stabilizes Aka at tSJ. Those results unravel a previous unknown role of M6 at tSJ and a tight interplay between tSJ components to assemble and maintain tSJs. In addition, tSJ components are not only essential at vertex as we found that loss of tSJ integrity also induces micron-length bicellular SJs deformations that are free of tensile forces. This phenotype is associated with the disappearance of SJ components at tricellular contacts, indicating that bSJ are no longer connected to tSJs. Reciprocally, SJ components are in turn required to restrict the localization of Aka and Gli at vertex. We propose that tSJs function as pillars to anchor bSJs to ensure the maintenance of tissue integrity in Drosophila proliferative epithelia.