Analysis of Gliotactin mediated microtubule organization during tricellular junction development

上皮细胞已经进化到保护下层组织免受各种和不断变化的环境影响。它们通过形成特殊的连接来保护组织的完整性，作为细胞旁扩散屏障。脊椎动物的上皮形成紧密连接，而无脊椎动物的上皮形成分离连接。脊椎动物和无脊椎动物的所有上皮的一个重要特征是在三个细胞聚集的地方存在专门的连接，称为三细胞连接。到目前为止，人们对这些结构、它们的组织和发育知之甚少，尽管它们的存在对于双细胞连接的正确发育是必要的。在模型无脊椎动物黑腹果蝇（Drosophila melanogaster）中，胶质动蛋白（Gliotactin）在三细胞连接处积累，是上皮完整性所必需的。到目前为止，其在三细胞连接处的超微结构定位及其功能尚不清楚。有趣的是，宿主实验室最近的研究表明，Gliotactin将会聚的分离连接链与三细胞连接处的微管连接起来。gliotactin依赖性微管动力学、组织和稳定性的分析代表了三细胞连接发展研究的一个未开发的领域。为了解决三细胞连接的组织和发展，我提出了一种结合光学和电子显微镜的方法，基于果蝇翅盘上皮。微管动力学在三细胞结发展将遵循在现场成像实验。随后，我将使用相关光学和电子显微镜（CLEM）来揭示微管，分离连接和三细胞塞之间的联系。为了分析微管在结形成中的功能作用，我将通过释放选择性药物或遗传工具来调节微管动力学并进行生理测量。此外，TEM断层扫描，聚焦离子束扫描电镜和免疫TEM将有助于破译三细胞连接的组织。最后，Gliotactin的新相互作用伙伴的识别将为Gliotactin如何促进三细胞连接的发展提供新的见解。

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.