The transmembrane proteins M6 and Anakonda cooperate to initiate tricellular junction assembly in epithelia ofDrosophila

Abstract

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

Acute manipulation and real-time visualization of membrane trafficking and exocytosis inDrosophila

“…Building on these findings, we employed RUSH to investigate the assembly of tricellular junctions (TCJs). The transmembrane proteins Anakonda (Aka), Gliotactin (Gli) and M6 accumulate at epithelial cell vertices, where they organize TCJs essential for epithelial barrier function (Schulte et al, 2003; Byri et al, 2015; Wittek et al, 2020), but how TCJ proteins are targeted to vertices is not clear. Analyzing the spatiotemporal dynamics of TCJ assembly has not been possible thus far, because recruitment of TCJ proteins to cell vertices is not apparent under steady-state conditions.…”

“…Flies carrying the homozygous viable Gli CPTI002805 allele (Lye et al, 2014) produce functional Gli::SVS protein that localized to TCJs like wild-type Gli protein (Fig. 6A; Wittek et al, 2020). Expression of two copies of ST-KDEL in epidermal stripes of heterozygous Gli CPTI002805 /+ embryos led to ER retention of Gli::SVS with only residual signals at TCJs, while Gli::SVS localized to TCJs in adjacent control cells (Fig.…”

“…Interestingly, the newly released Gli::SVS protein was not incorporated along the entire length of TCJs, but first accumulated at the apical side of pre-existing TCJs (Fig. 6C), suggesting that growing TCJs are extended by addition of new material to the apical side of the junctional complex, resembling growing SJs (Wittek et al, 2020; Babatz et al, 2018). These findings demonstrate how synchronization of membrane trafficking can be employed to gain insights into the spatiotemporal dynamics of junctional complex assembly in developing tissues in vivo .…”

Attachment/detachment of cortical myosin regulates cell junction exchange during cell rearrangement

“…To test this hypothesis, we performed loss-of-function experiments on TCJ proteins. We found that RNAi of m6, which encodes a component of the tricellular SJ (tSJ) [23][24][25] , increased the fraction of long-lasting rsMCs, without inducing the drastic malformation of the rsMC (Fig. 3b-e, Supplementary Fig.…”

“…It has been reported that M6 and the tSJ protein Anakonda (Aka) localize to the TCJ in a mutually dependent manner and that they cooperate in the TJC assembly in other epithelial tissues 24,26 . Thus, it is possible that Aka works with M6 in the junction exchange in the wing.…”