Basis and Function of Lateral Assembly of Cadherin Molecules in Adhesive Junctions of Humans and Model Organisms

人类和模型生物粘附连接中钙粘蛋白分子横向组装的基础和功能

• 批准号: 10715056
• 负责人: Julia Brasch
• 金额: $ 38.5万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-20 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10715056
• 关键词: Actins; Adherens Junction; Adhesives; Affinity; Animals; Architecture; Autoimmune Diseases; Binding; Biological Models; Biophysics; Bulla; Cadherins; Caenorhabditis elegans; Cell Separation; Cells; Cryoelectron Microscopy; Cytoskeleton; Data; Desmosomes; Development; Disease; Drosophila genus; Endothelium; Environment; Functional disorder; Genetic; Higher Order Chromatin Structure; Human; In Situ; Intercellular Junctions; Intermediate Filaments; Lateral; Link; Liposomes; Mechanical Stress; Membrane; Methods; Molecular; Morphogenesis; Mutagenesis; Mutation; Physiology; Property; Proteins; Resistance; Resolution; Skin; Solid; Specificity; Structure; System; Testing; Time; Tissues; Translating; Validation; Visualization; Whole Organism; X-Ray Crystallography; adhesion receptor; biophysical techniques; cost; extracellular; human disease; member; membrane reconstitution; model organism; mouse model; prevent; reconstitution; tumor progression

PROJECT SUMMARY / ABSTRACT Adherens junctions, desmosomes and endothelial junctions are fundamental adhesive junctions crucial to animal physiology. Mutations and autoimmune diseases targeting them cause severe disorders in humans. Adherens junctions are found in all solid tissues and link actin cytoskeletons of adjacent cells, while desmosomes form strong linkages between intermediate filaments to provide resistance to mechanical stress. Endothelial junctions are distinct, specialized adherens junctions that maintain the integrity of vessels. Members of the cadherin superfamily of Ca2+-dependent adhesion receptors form the core transmembrane components of each of these junctions. Remarkably, our preliminary data show that cadherins of desmosomal and endothelial junctions spontaneously form highly ordered, intricate junctional architectures between reconstituted membranes through adhesive trans interactions and putative lateral cis interactions between cadherins on the same membrane. We have previously characterized their adhesive trans interactions in detail, but the mechanisms by which they assemble ordered extracellular architectures, including the identity of lateral interfaces, remain unknown. Two projects in this proposal aim to illuminate this potentially critical, but poorly understood level of structural organization in molecular detail using a cryo-ET approach in reconstituted and native junctions. At the core of our approach is a method I developed to overcome the difficulties of studying lateral interactions, which tend to be weak and poorly detectable outside of a membrane environment. Purified ectodomains are used to reconstitute junctions on liposomes for direct visualization of assemblies using cryo-ET, providing domain-level resolution of these large structures and identifying specific interfaces for functional validation by mutagenesis. We will employ this system to determine the assembly and lateral interactions of vertebrate desmosomal and endothelial junctions, then extend our findings to native cellular junctions using in situ cryo-ET. Two further projects investigate how cadherin adhesive and lateral interaction properties translate into function at the level of tissue organization, integrity and cell sorting using the power of model organism genetics. Direct assessment of the effects of modifying cadherin properties on morphogenesis in whole organisms has been prevented by functional redundancy and the high cost, difficulty and time of mouse models. These barriers are overcome in model organisms, Drosophila and C. elegans, which have highly restricted cadherin repertoires and far superior genetic tractability. We aim to define the molecular interactions of the adherens junction cadherins of these model organisms by a combined x-ray crystallography, cryo-EM, cryo-ET and biophysical approach to open up the use of these facile model systems for structure-function studies testing mutations that modify adhesive binding specificities, affinities and lateral interactions. Overall, our approach will define a new level of junctional organization across a range of fundamental adhesive structures and provide a substantial step in our ability to interrogate the functional effects of cadherin molecular properties on tissue morphogenesis and architecture.

项目摘要 /摘要 粘附连接，脱糖体和内皮连接是对动物至关重要的基本粘合剂连接 生理。针对它们的突变和自身免疫性疾病引起人类严重疾病。附着力 在所有固体组织中都发现了连接，并将相邻细胞的肌动蛋白细胞骨架联系起 中间细丝之间的紧密联系以提供对机械应力的抗性。内皮连接 是保持船舶完整性的独特，专门的粘附连接。钙粘蛋白的成员 Ca2+依赖性粘附受体的超家族形成了每一种的核心跨膜成分 连接。值得注意的是，我们的初步数据表明，脱粒和内皮结的钙粘蛋白 自发地形成高度有序的，复杂的连接架构，通过重构膜之间 在同一膜上钙粘着蛋白之间的粘合性反式相互作用和推定的侧面顺式相互作用。我们 以前已经详细介绍了它们的粘合性反式相互作用，但是它们的机制 组装有序的细胞外体系结构，包括横向界面的身份，仍然未知。二 该提案中的项目旨在阐明这种潜在的至关重要但知之甚少的结构水平 在重构和天然连接中使用冷冻-ET方法进行分子细节的组织。核心 我们的方法是我开发的一种方法来克服研究横向相互作用的困难，这往往 在膜环境之外弱且无法检测到。纯化的外域用于 在脂质体上重建连接处，用于使用Cryo-Et直接可视化组件，提供域级 这些大型结构的分辨率，并识别通过诱变验证功能验证的特定界面。 我们将采用该系统来确定脊椎动物载液和侧面相互作用 内皮连接，然后使用原位冷冻-ET扩展到本地细胞连接。另外两个 项目研究钙粘蛋白粘合剂和横向相互作用的特性如何转化为级别的功能 使用模型生物遗传学的力量，组织组织，完整性和细胞分类。直接评估 修饰钙粘蛋白特性对整个生物体形态发生的影响已被阻止 功能冗余以及小鼠模型的高成本，困难和时间。这些障碍被克服 模型有机体，果蝇和秀丽隐杆线虫，它们具有高度限制的钙粘蛋白曲目，并且优越得多 遗传性障碍。我们旨在定义这些粘附素连接蛋白的分子相互作用 通过组合的X射线晶体学，冷冻EM，冷冻-ET和生物物理方法的模型生物可以打开 这些便利模型系统用于结构功能研究测试突变，以修饰粘合剂 结合特异性，亲和力和横向相互作用。总体而言，我们的方法将定义一个新的连接水平 组织各种基本的粘合剂结构，并为我们的能力提供了重要的一步 询问钙粘蛋白分子特性对组织形态发生和结构的功能作用。