Role of E-cadherin’s N-glycans in tissue morphogenesis

进化保守的E-钙粘蛋白(果蝇中的猎枪)是上皮细胞中一种细胞黏附分子的原型，它一方面负责同型的和钙依赖的细胞与细胞之间的黏附，另一方面通过与β-连环蛋白(果蝇中的瓢虫)和α-连环蛋白形成三聚体复合体与皮质肌球蛋白细胞骨架相连。除了这些普通的三聚体复合体外，E-钙粘附素还存在于数百个分子的超分子复合体中，它们可能参与也可能不参与粘附性和三聚体复合体。E-钙粘蛋白位于上皮细胞间的力传递的中心位置，在机械细胞通讯中起着关键作用。由于E-钙粘附素在黏附、皮质连接和机械转导中的作用相互交织，其分子机制一直难以研究。基于低-N-糖基化的E-钙粘蛋白在聚集、皮质相互作用和细胞-细胞通讯的某些方面受到损害的发现，我们建议系统地研究N-糖链在果蝇胚胎组织形态发生过程中通过N-糖基化位点的一系列E-钙粘附素突变在生理环境中对E-钙粘蛋白功能的贡献。这些突变的表型将通过建立的E-钙粘蛋白迁移率、复合体形成、连接机制、黏附、细胞信号转导等方面的已建立的分子和功能分析来研究。纳米结构中的表型将通过高分辨率显微镜和DNA-PAINT单分子定位来研究。

The evolutionary conserved E-cadherin (shotgun in Drosophila) is the prototype of a cell adhesion molecule in epithelia, which on the one side is responsible for homotypic and Calcium dependent cell-cell adhesion and on the other is connected to the cortical actomyosin cytoskeleton by forming a trimeric complex with beta-catenin (armadillo in Drosophila) and alpha-catenin. Beside such generic trimeric complexes, E-cadherin is found in super-molecular complexes of several hundred molecule, which may or may not be engaged in adhesion and trimeric complexes. Being centrally positioned at the force transmission between epithelial cells, E-cadherin plays a pivotal role in mechanical cell communication. The molecular mechanism of mechanotransduction has been difficult to investigate given the intertwined E-cadherin functions in adhesion, cortical link and mechanotransduction. Based on findings that hypo-N-glycosylated E-cadherin is impaired in clustering, cortical interactions and certain aspects of cell-cell communication, we propose to systematically investigate the contributions of N-glycans to the functions of E-cadherin in a physiological setting during tissue morphogenesis in Drosophila embryos by a series of E-cadherin mutations at N-glycosylation sites. The phenotype of these mutations will be investigated by established molecular and functional assays for E-cadherin mobility, complex formation, mechanics of junctions, adhesion, cell signalling. The phenotypes in nano-structure will be investigated by high resolution microscopy and DNA-paint single molecular localization.