Palmitoylation of proteolipid protein M6 promotes tricellular junction assembly in epithelia of Drosophila.

Tricellular junctions (TCJs) seal epithelial cell vertices and are essential for tissue integrity and physiology, but how TCJs are assembled and maintained is poorly understood. In Drosophila, the transmembrane proteins Anakonda (Aka, also known as Bark), Gliotactin (Gli) and M6 organize occluding TCJs. Aka and M6 localize in an interdependent manner to vertices and act jointly to localize Gli, but how these proteins interact to assemble TCJs was not previously known. Here, we show that the proteolipid protein M6 physically interacts with Aka and with itself, and that M6 is palmitoylated on conserved juxta-membrane cysteine residues. This modification promotes vertex localization of M6 and binding to Aka, but not to itself, and becomes essential when TCJ protein levels are reduced. Abolishing M6 palmitoylation leads to delayed localization of M6 and Aka but does not affect the rate of TCJ growth or mobility of M6 or Aka. Our findings suggest that palmitoylation-dependent recruitment of Aka by M6 promotes initiation of TCJ assembly, whereas subsequent TCJ growth relies on different mechanisms that are independent of M6 palmitoylation.

Acute manipulation and real-time visualization of membrane trafficking and exocytosis in Drosophila.

Intracellular trafficking of secretory proteins plays key roles in animal development and physiology, but so far, tools for investigating the dynamics of membrane trafficking have been limited to cultured cells. Here, we present a system that enables acute manipulation and real-time visualization of membrane trafficking through the reversible retention of proteins in the endoplasmic reticulum (ER) in living multicellular organisms. By adapting the "retention using selective hooks" (RUSH) approach to Drosophila, we show that trafficking of GPI-linked, secreted, and transmembrane proteins can be controlled with high temporal precision in intact animals and cultured organs. We demonstrate the potential of this approach by analyzing the kinetics of ER exit and apical secretion and the spatiotemporal dynamics of tricellular junction assembly in epithelia of living embryos. Furthermore, we show that controllable ER retention enables tissue-specific depletion of secretory protein function. The system is broadly applicable to visualizing and manipulating membrane trafficking in diverse cell types in vivo.

Src42A is required for E-cadherin dynamics at cell junctions during Drosophila axis elongation.

Src kinases are important regulators of cell adhesion. Here, we have explored the function of Src42A in junction remodelling during Drosophila gastrulation. Src42A is required for tyrosine phosphorylation at bicellular (bAJ) and tricellular (tAJ) junctions in germband cells, and localizes to hotspots of mechanical tension. The role of Src42A was investigated using maternal RNAi and CRISPR-Cas9-induced germline mosaics. We find that, during cell intercalations, Src42A is required for the contraction of junctions at anterior-posterior cell interfaces. The planar polarity of E-cadherin is compromised and E-cadherin accumulates at tricellular junctions after Src42A knockdown. Furthermore, we show that Src42A acts in concert with Abl kinase, which has also been implicated in cell intercalations. Our data suggest that Src42A is involved in two related processes: in addition to establishing tension generated by the planar polarity of MyoII, it may also act as a signalling factor at tAJs to control E-cadherin residence time.

The Transmembrane Proteins M6 and Anakonda Cooperate to Initiate Tricellular Junction Assembly in Epithelia of Drosophila.

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-transmembrane-domain 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 low-mobility 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.