Defining the mechanisms that remodel the multivalent domain structures of cell junctions during morphogenetic activities

在我的项目中，我将进一步了解细胞在形态发生过程中如何保持彼此连接，在形态发生过程中，连接复合体必须对细胞形状变化和有丝分裂活动做出动态反应。这一过程的失败可能会导致癌细胞发育障碍和转移。我假设这种动态反应是基于具有多价结构域的连接复合体的结构，该结构域调节连接到细胞骨架的连接。在本研究中，我将进一步研究连接相关蛋白PYD和CNO在果蝇胚胎上皮组织中的作用。果蝇胚胎发育过程中的生殖带延长和背部关闭过程为研究连接动力学提供了有用的工具，因为它们是由细胞嵌入、有丝分裂和组织运动所产生的高组织力所定义的。在CNO和PYD突变胚胎中，尤其在组织应力升高的区域观察到组织破裂和细胞形状扰动。因此，这些蛋白质似乎通过能够将连接复合体连接到细胞骨架来提供对附着连接的稳健性。然而，它们在这一过程中的机械作用仍不清楚。我将通过对突变表型的详细研究来分析PYD对连接稳健性的影响，并研究已知的PYD相互作用因子在这一过程中的作用。初步数据可以证实PYD突变体在生殖带延长过程中的扰动细胞形状，这可能是扰动细胞嵌入和连接动力学的结果。对固定和活标本的进一步研究将包括对细胞形状、细胞嵌入和连接动力学的详细描述和量化，这将有助于了解这些连接相关蛋白的功能。组织中的细胞是动态的，这要求动态连接必须确保有丝分裂、细胞插入和细胞形状变化期间的完整性。因此，稳定的细胞-细胞连接一方面应该提供刚性，但另一方面应该对变化做出灵活的反应。因此，我假设连接复合体及其与细胞骨架的连接显示了相互作用的蛋白质的亚结构，从而提供了提供这两种需求的能力。潜在亚结构的主要迹象是成熟果蝇上皮中连接相关蛋白ARM、CNO和BAZ的部分分离。在我的研究中，我将利用超分辨率显微镜进一步剖析果蝇胚胎和哺乳动物细胞中细胞连接的亚结构。这两个系统都将研究它们成熟和维护过程中的连接动力学。此外，我将比较一个组织中表现出差别力的连接的亚结构，例如，具有更高力的三细胞连接与双细胞连接。

In my project I will gain further understanding how cells remain attached to each other during morphogenetic processes in which the junctional complexes have to respond dynamically to cell shape changes and mitotic activity. A failure of this process can lead to developmental disorders and metastasis of cancer cells. I hypothesize that this dynamic response is based on the structure of the junctional complex with multivalent domains that regulate the connection of junctions to the cytoskeleton. In this study, I will further investigate the role of the junction-associated proteins Pyd and Cno in embryonal epithelial tissues of Drosophila melanogaster. The processes of germ band elongation and dorsal closure during Drosophila embryogenesis provide a useful tool to investigate junctional dynamics as they are defined by high tissue forces due to cell intercalation, mitosis and tissue movement. In cno and pyd mutant embryos, tissue rupture and perturbed cell shapes were especially observed at regions with elevated tissue stress. Thus, these proteins seem to provide robustness to adherens junctions by being able to connect the junctional complex to the cytoskeleton. However, their mechanistical role in this process remained unclear. I will analyze the effect of Pyd on junction robustness by detailed examination of mutant phenotypes and investigate the roles of known Pyd interactors in this process. Preliminary data could confirm perturbed cell shapes in pyd mutants during the process of germ band elongation that could be a result of perturbed cell intercalation and junction dynamics. Further investigation of fixed and live specimen will include detailed description and quantification of cell shapes, cell intercalation and junction dynamics which will help to understand the function of these junction-associated proteins.The fact that cells within a tissue are dynamic requires dynamic junctions which have to ensure integrity during mitosis, cell intercalation and cell shape changes. Thus, the stable cell-cell junction should on the one hand provide stiffness but on the other hand respond flexible on changes. Thus, I hypothesize that the junctional complex and its connection to the cytoskeleton display a substructure of interacting proteins that provide the ability to provide both requirements. Primary indication of a potential substructure is the partial segregation of the junction-associated proteins Arm, Cno and Baz in mature Drosophila epithelia. During my study, I will further dissect the substructure of cell junctions in Drosophila embryos and mammalian cells by super-resolution microscopy. In both systems will study junction dynamics during their maturation and maintenance. Furthermore, I will compare the substructures of junctions within one tissue that exhibit differential forces, f. e. tricellular junctions with higher forces versus bicellular junctions.