Vertebrate tight junctions (TJs) and septate junctions (SJs) in invertebrates are diffusional barriers between adjacent cells and are essential for epithelial homeostasis. Specialized TJs and SJs, called tricellular junctions (TCJs), are found at vertices where three cells meet, and are structurally unique from other junctions. In Drosophila, TCJs are formed by a protein complex of Anakonda (Aka), Gliotactin (Gli) and M6. Aka and M6 localize to TCJs in a mutually dependent fashion and recruit Gli; however, how these proteins interact to assemble TCJs is unclear. In this study (Schleutker and Luschnig, 2024), the authors demonstrate that M6, like its vertebrate homologue GPM6a, is S-palmitoylated in vivo, and that this lipid modification is attached to conserved juxta-membrane cysteine residues. By generating a palmitoylation-deficient mutant, they show that although palmitoylation is required for correct timing of M6 and Aka enrichment at TCJs, it is not required for subsequent TCJ growth. However, M6 palmitoylation becomes essential when Aka levels are reduced, leading to significantly attenuated recruitment of Gli to TCJs, and resulting in embryonic lethality. Mechanistically, the authors find that the palmitoyl modification mediates M6 binding to Aka but is not required for M6–M6 interactions. Overall, this study shows that M6 palmitoylation is required for the initial assembly of TCJs and provides interesting insights into the molecular requirements for TCJ formation.
