Analysis of Gliotactin mediated microtubule organization during tricellular junction development

上皮细胞已经进化，以保护底层组织免受不同和不断变化的环境影响。它们通过形成特殊的连接来起到细胞旁扩散屏障的作用，以保持组织的完整性。脊椎动物的上皮细胞形成紧密的连接，而无脊椎动物则形成分隔的连接。脊椎动物和无脊椎动物的所有上皮细胞的一个重要特征是在三个细胞汇合的点上存在专门的连接，称为三细胞连接。到目前为止，人们对这些结构及其组织和发育知之甚少，尽管它们的存在是正确发展双细胞连接所必需的。在无脊椎动物黑腹果蝇的模型中，Gliotactin蛋白聚集在三细胞连接处，是上皮完整性所必需的。到目前为止，它在三细胞连接处的超微结构定位及其功能仍不清楚。有趣的是，来自宿主实验室的最新工作表明，Gliotactin将会聚的隔状连接链连接到三细胞连接处的底层微管。分析依赖于Gliotactin的微管的动力学、组织和稳定化是三细胞连接发育研究的一个未知领域。为了研究三细胞连接的组织和发育，我提出了一种基于光镜和电子显微镜相结合的方法，在果蝇的翼盘上皮细胞中。在活体成像实验中，将跟踪三细胞连接发育过程中的微管动力学。随后，我将使用CLEM(相关光学和电子显微镜)来揭示微管、分隔连接和三细胞栓之间的联系。为了分析微管在连接形成中的功能作用，我将通过选择性药物或遗传工具的去掉来调节微管的动力学，并进行生理测量。此外，透射电子显微镜断层扫描、聚焦离子束扫描和免疫电子显微镜将有助于破译三细胞连接的组织。最后，Gliotactin新的相互作用伙伴的发现将为Gliotactin如何促进三细胞连接的发育提供新的见解。

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.