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Biochemical protein interactions of Gliotactin at the tricellular junction Que, Jaimmie

Abstract

The septate junction is an occluding junction in invertebrates, similar in function to tight junctions, playing a role in epithelial barriers, and in apical/basal polarity. Septate junction interactions are still being characterized as new component proteins are discovered. One septate junction protein, Gliotactin, was discovered in Drosophila to correlate with the mislocalization of characteristic septate junction proteins in a Gliotactin null animal. However, Gliotactin is the only component found exclusively at the level of septate junctions at tricellular junctions in epithelia. The tricellular junctions are the structure at the convergence of three cells and a potential organizing factor of the septate junction. This led to the question, what is Gliotactin's role in the organization of the tricellular and septate junctions? To study this, we looked at Gliotactin interactors, and attempted to elucidate a model of tricellular and septate junction protein interactions. Previous attempts at finding Gliotactin interactors were made using in vitro systems or by using transgenic animals using an over-expressed epitope-tagged Gliotactin, that showed that Gliotactin interacts with known septate junction proteins in a calcium dependent manner. This thesis aims to further explore Gliotactin interactions with the hypothesis that native Gliotactin in Drosophila interacts in a calcium dependant manner with septate junction proteins, Discs Large, and Neurexin IV. Using co-immunoprecipitation and GST pulldowns, on native Gliotactin protein in Drosophila, I have shown that Gliotactin does not interact with Neurexin IV but does interact with Discs Large in a calcium-dependent manner. This is significant in that, to date, there has been no known interactor of Discs Large or of native Gliotactin at the tricellular junction. I also present data on unidentified potential Gliotactin interactors seen in a GST pulldown assay. The data presented in this thesis has contributed to a new working model of the tricellular junction and the role of Gliotactin.

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