Strategies to identify Gliotactin protein interactors in Drosophila

Gliotactin is a transmembrane choline-esterase-like molecule required for the transepithelial and blood-nerve barriers in Drosophila. In epithelia, Gliotactin is the only known marker of cell corners, and is required for both parallel wing hair alignment and septate junction maturation. These functions infer Gliotactin association (direct or indirect) with septate junction components and cytoskeletal factors which, with exception to Discs Large, have yet to be identified. For this reason, a number of strategies were employed to identify protein interactors with Gliotactin, yielding several promising results. Firstly, in immunoprecipitates, Gliotactin was phosphorylated at two conserved tyrosine residues within its C-terminus. One or both of these residues was shown to be phosphorylated in vitro by Src Kinase, a process which could possibly be related to Gliotactin recycling/degradation. In addition, through application of immunoprecipitations in combination with LC-MS/MS analysis, a promising method was developed for future identification of protein interactions. Of the proteins identified in the course of this work, 14-3-3 isoforms and the Na⁺/K⁺ ATPase were shown to colocalize with septate junctions, with the Na⁺/K⁺ ATPase showing strong correlation with both Gliotactin and Discs Large. 14-3-3 also had spot concentrations between dividing cells, possibly reflecting a role in cytokinesis which has been recently described in mammals. This study presents several possible targets for pursuit as both Gliotactin interactors, and as possible modulators of cytokinesis (14-3-3) and Glial adhesion (Magi). The methods described form a model by which future identification of novel interactors and other biochemical studies can be designed.Science, Faculty ofZoology, Department ofGraduat