Analysis of Gliotactin mediated microtubule organization during tricellular junction development

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Epithelial cells have evolved to shield the underlying tissue from diverse and changing environmental influences. They act as paracellular diffusion barrier by forming specialized junctions that preserve tissue integrity. Vertebrate epithelia form tight junctions, whereas invertebrates establish septate junctions. An important feature of all epithelia in both vertebrates and invertebrates is the presence of specialized junctions at the point where three cells converge, known as tricellular junctions. So far, very little is known about these structures, their organization and their development, although their presence is necessary for the correct development of bicellular junctions.In the model invertebrate Drosophila melanogaster, the protein Gliotactin accumulates at tricellular junctions and is required for epithelial integrity. So far, its ultrastructural localization at the tricellular junction and its function are still unclear. Intriguingly, recent work from the host lab suggests that Gliotactin links the converging septate junctions strands to the underlying microtubules at the tricellular junction. The analysis of Gliotactin-dependent microtubule dynamics, organization and stabilization represents an unexplored area of tricellular junction development research.To address the organization and development of tricellular junctions I propose a combined light and electron microscopy based approach in the wing disc epithelium of Drosophila. Microtubule dynamics during tricellular junction development will be followed in live imaging experiments. Subsequently I will use CLEM (correlative light and electron microscopy) to uncover the connection between microtubules, septate junctions and tricellular plugs. To analyze the functional roles of microtubules in junction formation I will modulate microtubule dynamics by uncaging of selective drugs or genetic tools and perform physiological measurements. Moreover, TEM tomography, Focused Ion Beam SEM and immuno-TEM will help to decipher the organization of tricellular junctions. Finally, the identification of novel interaction partners of Gliotactin will give new insights into how Gliotactin contributes to tricellular junction development.