Gliotactin and Discs large form a protein complex at the tricellular junction of polarized epithelial cells inDrosophila

Abstract

The tricellular junction (TCJ) forms at the convergence of pleated septate junctions (SJs) from three adjacent cells in polarized epithelia and is necessary for maintaining the transepithelial barrier. In Drosophila, the transmembrane protein Gliotactin was the first identified marker of the TCJ, but little is known about other molecular constituents. We now show that Gliotactin associates with Discs large at the TCJ in a Ca2+-dependent manner. Discs large is essential for the formation of the TCJ and the localization of Gliotactin. Surprisingly, Gliotactin localization at the TCJ was independent of its PDZ-binding motif and Gliotactin did not bind directly to Discs large. Therefore Gliotactin and Discs large association is through intermediary proteins at the TCJ. Gliotactin can associate with other septate junction proteins but this was detected only when Gliotactin was overexpressed and spread throughout the septate junction domain. Gliotactin overexpression and spread also resulted in a reduction of Discs large staining but not vice versa. These results suggest that Discs large participates in different protein interactions in the SJ and the TCJ. Finally this work supports a model where Gliotactin and Dlg are components of a larger protein complex that links the converging SJs with the TCJ to create the transepithelial barrier.

Identification of the septate junction protein gliotactin in the mosquito, Aedes aegypti: evidence for a role in increased paracellular permeability in larvae

“…We hypothesized that Gli would localize to the TCJ complex in the epithelia of larval mosquito, as has previously been reported in D. melanogaster (see Schulte et al, 2003Schulte et al, , 2006; however, this was not the case as in most tissues examined, Gli localized at bicellular SJs. In contrast, the hypothesis that Gli expression would respond to alterations in salinity can be accepted as Gli levels were found to exhibit an organ-specific increase in animals reared in BW.…”

“…The vertebrate homologs of Gli, neuroligins, have been shown to dimerize or oligomerize via their extracellular serine esterase-like domain (Ichtchenko et al, 1995(Ichtchenko et al, , 1996, and Gli dimerization has also been suggested for D. melanogaster (Venema et al, 2004;PadashBarmchi et al, 2013). In contrast, we can not rule out the possibility that the observed ∼245 kDa Gli band may be a product of a Gli heterodimer complex with another SJ protein such as Dlg, which has been shown to occur in adult D. melanogaster (Schulte et al, 2006).…”

“…This observation is inconsistent with D. melanogaster Gli, which is reported to be concentrated at the TCJs in the midgut epithelium (Byri et al, 2015). However, in the epithelia of epidermis and wing imaginal discs, D. melanogaster Gli spreads throughout the entire SJ domain and interacts with other bicellular SJ components when overexpressed (Schulte et al, 2006). As mentioned above, the presence of Gli at the bicellular SJs in D. melanogaster epithelia results in tissue overgrowth and apoptosis (Padash-Barmchi et al, 2010.…”

“…2A). The expression of Gli in a wide range of epithelial tissues, including the midgut and hindgut, has been shown in larval D. melanogaster (Schulte et al, 2003(Schulte et al, , 2006Byri et al, 2015). In contrast, Gli is not expressed in the D. melanogaster Malpighian tubules (Schulte et al, 2006).…”

“…In polarized epithelia, Gli levels and its TCJ localization are tightly regulated via phosphorylation, endocytosis and degradation (Padash-Barmchi et al, 2010). When overexpressed, Gli spreads away from the TCJ into the bicellular SJ domain, where it interacts with the cytoplasmic SJ protein discs large (Dlg) (Schulte et al, 2006;Padash-Barmchi et al, 2010. Gli interaction with Dlg results in reduced Dlg levels, tissue overgrowth and apoptosis (Schulte et al, 2006;Padash-Barmchi et al, 2010.…”