Structural Studies of Type II Cadherins on Membranes by Cryo-Electron Tomography

项目摘要 细胞-细胞连接控制细胞粘附，并以跨膜粘附蛋白阵列为特征 结合在两个并列细胞的质膜之间。大量的细胞间连接，包括紧密的 连接、缝隙连接、突触等在脊椎动物中是保守的，在生物学上起着至关重要的作用。 然而，尽管其核心重要性，目前还没有令人满意的方法来调查其 详细的三维结构。在这里，我提出了新的方法来重建和成像细胞-细胞 通过冷冻电子断层扫描（cryo-ET），我将开发和验证钙粘蛋白介导的 细胞间连接钙粘蛋白细胞粘附蛋白提供桥粒的细胞间附着 和粘附连接。这些连接处的钙粘蛋白自组装形成致密的分子晶格 依赖于钙粘蛋白胞外域之间的特异性顺式（相同细胞）和反式（并置细胞）相互作用。我 将研究由两种类型的钙粘蛋白产生的连接：血管内皮（VE）钙粘蛋白，它形成 血管内皮连接和钙粘蛋白-11，其通常表达为 在伤口愈合过程中，其过度表达可导致类风湿性关节炎。我将制作 使用脂质体或大的扁平脂质纳米盘重建与每种蛋白质的连接，并使用冷冻ET 以确定组装接头的结构。我也将继续发展我的冷冻ET管道， 采用了一种新颖的无基准标记的方法来处理图像数据。我将提供由此产生的技术 冷冻ET方法和软件的科学界，从而扩大了这一研究项目的影响。 总的来说，该项目预计将产生第一个详细的内皮连接的3D结构， VE-钙粘蛋白，和由II型钙粘蛋白，钙粘蛋白-11形成的连接的第一结构。我预计这 该方法通常适用于细胞-细胞连接的研究，为许多未来的研究开辟了可能性。 问题研究

Project Summary Cell-cell junctions control cell adhesion and are characterized by arrays of transmembrane adhesion proteins bound between the plasma membranes of two apposed cells. Numerous cell-cell junctions, including tight junctions, gap junctions, synapses, and others, are conserved among vertebrates and play crucial biological roles, yet despite their central importance, there are currently no satisfactory methods to investigate their detailed three-dimensional structures. Here I propose new methods for reconstituting and imaging cell-cell junctions by cryo-electron tomography (cryo-ET), which I will develop and validate with cadherin-mediated intercellular junctions. Cadherin cell adhesion proteins provide the intercellular attachments of desmosomes and adherens junctions. Cadherins of these junctions self-assemble to form dense molecular lattices dependent on specific cis (same cell) and trans (apposed cell) interactions between cadherin ectodomains. I will study the junctions produced by two types of cadherins: vascular endothelial (VE) cadherin, which forms the well-characterized endothelial junctions of blood vessels, and cadherin-11, which is normally expressed during wound healing, and for which over-expression can lead to rheumatoid arthritis. I will produce reconstituted junctions with each protein using either liposomes or large, flat lipid nanodiscs, and use cryo-ET to determine the structures of the assembled junctions. I will also continue to develop my cryo-ET pipeline that employs a novel, fiducial marker-free method for processing image data. I will supply the resulting technical cryo-ET methods and software to the scientific community, thus widening the impact of this research project. Overall, this project is expected to produce the first detailed 3D structures of endothelial junctions produced by VE-cadherin, and the first structures of a junction formed by a type II cadherin, cadherin-11. I expect this method to be generally applicable to the study of cell-cell junctions, opening possibilities for many future studies.