Cell vertices in epithelia comprise specialized tricellular junctions (TCJs) that seal the paracellular space between three adjoining cells [1,2]. Although TCJs play fundamental roles in tissue homeostasis, pathogen defense, and in sensing tension and cell shape [3-5], how they are assembled, maintained and remodeled is poorly understood. In Drosophila the transmembrane proteins Anakonda (Aka [6]) and Gliotactin (Gli [7]) are TCJ components essential for epithelial barrier formation. Additionally, the conserved four-transmembranedomain protein M6, the only myelin proteolipid protein (PLP) family member in Drosophila, localizes to TCJs [8,9]. PLPs associate with cholesterol-rich membrane domains and induce filopodia formation [10,11] and membrane curvature [12], and Drosophila M6 acts as a tumor suppressor [8], but its role in TCJ formation remained unknown. Here we show that M6 is essential for the assembly of tricellular, but not bicellular occluding junctions, and for barrier function in embryonic epithelia. M6 and Aka localize to TCJs in a mutually dependent manner and are jointly required for TCJ localization of Gli, whereas Aka and M6 localize to TCJs independently of Gli. Aka acts instructively and is sufficient to direct M6 to cell vertices in the absence of septate junctions, while M6 is required permissively to maintain Aka at TCJs. Furthermore, M6 and Aka are mutually dependent for their accumulation in a lowmobility pool at TCJs. These findings suggest a hierarchical model for TCJ assembly, where Aka and M6 promote TCJ formation through synergistic interactions that demarcate a distinct plasma membrane microdomain at cell vertices. Results and Discussion M6 co-localizes with Aka and Gli at tricellular junctions in embryonic epitheliaWe analyzed the distribution of M6 in embryos using a GFP protein trap insertion into the M6 locus, yielding GFP-tagged variants of five of the six annotated M6 isoforms (M6 CA06602 , referred to as GFP::M6; Fig. 1A [13]), or using an antiserum raised against two peptides present in five M6 protein isoforms (Fig. S1A). Anti-M6 immunostaining detected M6 protein in epithelia, including epidermis, salivary glands, tracheae, hindgut, midgut, Malpighian tubules, as well as in the nervous system of stage 14 embryos (Fig. S1D-I). In the epidermis of stage 15 embryos, M6 was detectable at lateral cell membranes (Fig. S1B,C). Compared to anti-M6 staining, GFP::M6 CA06602 displayed stronger accumulation at TCJs (Fig. S1B), suggesting that anti-M6 preferentially detects M6 isoforms or trafficking intermediates that are not GFP-positive in GFP::M6 CA06602 embryos. GFP::M6 accumulated at vertices at the level of septate junctions (SJs) marked by ; Fig. 1B), and co-localized with the TCJ components Aka and Gli (Fig. 1C). In living embryos GFP::M6 was enriched 7.56 +/-0.66-fold (n=10) at vertices relative to bicellular contacts,
Wittek et al.2 SummaryCell vertices in epithelia comprise specialized tricellular junctions (TCJs) that seal the paracellular space between three adjoining cells [1,2]. Although TCJs play fundamental roles in tissue homeostasis, pathogen defense, and in sensing tension and cell shape [3][4][5], how they are assembled, maintained and remodeled is poorly understood. In Drosophila the transmembrane proteins Anakonda (Aka [6]) and Gliotactin (Gli [7]) are TCJ components essential for epithelial barrier formation. Additionally, the conserved four-transmembranedomain protein M6, the only myelin proteolipid protein (PLP) family member in Drosophila, localizes to TCJs [8,9]. PLPs associate with cholesterol-rich membrane domains and induce filopodia formation [10,11] and membrane curvature [12], and Drosophila M6 acts as a tumor suppressor [8], but its role in TCJ formation remained unknown. Here we show that M6 is essential for the assembly of tricellular, but not bicellular occluding junctions, and for barrier function in embryonic epithelia. M6 and Aka localize to TCJs in a mutually dependent manner and are jointly required for TCJ localization of Gli, whereas Aka and M6 localize to TCJs independently of Gli. Aka acts instructively and is sufficient to direct M6 to cell vertices in the absence of septate junctions, while M6 is required permissively to maintain Aka at TCJs. Furthermore, M6 and Aka are mutually dependent for their accumulation in a lowmobility pool at TCJs. These findings suggest a hierarchical model for TCJ assembly, where Aka and M6 promote TCJ formation through synergistic interactions that demarcate a distinct plasma membrane microdomain at cell vertices. Results and Discussion M6 co-localizes with Aka and Gli at tricellular junctions in embryonic epitheliaWe analyzed the distribution of M6 in embryos using a GFP protein trap insertion into the M6
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