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

项目总结/摘要 粘附连接、桥粒和内皮连接是动物重要的粘附连接 physiology.针对它们的突变和自身免疫性疾病会导致人类严重疾病。粘着 在所有实体组织中发现连接，连接相邻细胞的肌动蛋白细胞骨架，而桥粒形成 中间丝之间的强连接以提供对机械应力的抵抗力。内皮连接 是不同的，专门的粘附连接，维持血管的完整性。钙粘蛋白成员 Ca 2+依赖性粘附受体的超家族形成这些受体中的每一个的核心跨膜组分。 交叉点值得注意的是，我们的初步数据表明，桥粒和内皮连接的钙粘蛋白， 自发形成高度有序的，复杂的连接结构之间的重组膜，通过 同一膜上钙粘蛋白之间的粘附性反式相互作用和假定的侧向顺式相互作用。我们 先前已经详细描述了它们的粘合剂反式相互作用，但是它们的机制 组装有序的细胞外结构，包括横向接口的身份，仍然是未知的。两 本提案中的项目旨在阐明这一潜在的关键，但知之甚少的结构水平 在重组和天然连接中使用冷冻ET方法进行分子细节组织。为核心 我们的方法是我为了克服研究横向相互作用的困难而开发的一种方法，这种方法往往会 在膜环境之外是弱的和难以检测的。纯化的胞外域用于 在脂质体上重建连接，以便使用冷冻ET直接观察组装体， 这些大结构的解析和鉴定用于通过诱变进行功能验证的特定界面。 我们将利用这一系统来确定脊椎动物桥粒的组装和横向相互作用， 内皮连接，然后扩展我们的研究结果，天然细胞连接使用原位冷冻ET。两个另外 项目研究钙粘蛋白粘附和横向相互作用特性如何转化为水平功能 组织的组织性、完整性和细胞的分类，利用模式生物遗传学的力量。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， 功能冗余和鼠标模型的高成本、高难度和高时间。这些障碍被克服， 模式生物果蝇和C. elegans，其具有高度限制的钙粘蛋白库和远优于上级 遗传易处理性我们的目标是确定这些粘附连接钙粘蛋白的分子相互作用， 通过结合x射线晶体学，cryo-EM，cryo-ET和生物物理方法来建立模型生物， 使用这些简单的模型系统进行结构-功能研究，测试改变粘合剂的突变 结合特异性、亲和力和横向相互作用。总的来说，我们的方法将定义一个新的连接级别， 组织在一系列基本的粘合剂结构，并提供了一个实质性的一步，我们的能力， 询问钙粘蛋白分子特性对组织形态发生和结构的功能影响。

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.