LASP1 as a functional linker protein between cadherin and actin dynamics at epithelial adherens junctions

LASP1 作为上皮粘附连接处钙粘蛋白和肌动蛋白动力学之间的功能性连接蛋白

• 批准号: 414058881
• 负责人: Professor Dr. Hans-Joachim Schnittler
• 金额: --
• 依托单位: Institut für Anatomie und Vaskuläre Biologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/414058881?language=en
• 关键词: LASP1; functional; linker; protein; between

Epithelial cadherin (E-cadherin) mediates epithelial adhesion and connects via the beta-catenin/alpha-catenin complex to the actin cytoskeleton that in turn contributes to adherence junction organization and dynamics. Epithelia display an organ-specific Phenotype that is beyond tight junctions largely determine by adherens junctions. We postulate that the supramolecular organization of adherens junctions has a significant impact on the organization and function of AJs. Similarly, we suspect that the expression of mesenchymal cadherins such as VE-cadherin and cadherin-11 during EMT alters the supramolecular organization of AJs, thereby contributing to an altered cell-junction dynamics of carcinomas. Recently we found a novel mechanism of junction regulation in endothelium, which is based on actin-driven junction-associated intermittent lamellipodia (JAIL). JAIL drive VE-cadherin dynamics and thus controls adhesion features, cell migration and monolayer integrity. Similarily, here we show for the first time that epithelial cell lines and human breast carcinoma cell lines display comparable JAIL-like structures. Furthermore, AJ organization and dynamics is controlled by actin-binding and cadherin regulating proteins together with signaling molecules involving acto-myosin mediated contractility. In this context, we identified the LIM and SH3 protein 1 (LASP1), an actin binding protein, as a component of epithelial adherens junctions, and found it overexpressed in dedifferentiated breast cancer MDA cell lines as well as in chronically inflamed tissue, situations in which alterations in cell-to-cell adhesion plays a central role. Furthermore, we identified LASP1 as a new binding partner for β-catenin, which localizes at epithelial adherens junctions and interestingly in JAIL-like plasma membrane protrusion. Mechanistically, we propose LASP1 as a novel adherens junction associated protein in epithelium, which in concerted interaction with actin and b-catenin contributes to junction organization and dynamics. To test the impact of LASP1 and ectopically expressed cadherins on adherens junction dynamics, we will investigate different differentiated MDCK cell lines and human breast cancer cell lines that ectopic express cadherin-11 and VE-cadherin. Fluorescence tagged adherens junction and cytoskeletal proteins will be expressed and fluorescence-based life cell imaging performed to quantitatively monitor cadherin and actin dynamics. Data will be complemented by superresolution microscopy to characterize the supramolecular organization of adherens junctions in a cadherin and LASP1 dependent manner along with established cell- and molecular biology techniques. We plan to use a CHO expression system for confirmation of particular interactions. We expect to establish a molecular mechanistic model that helps to explain the differentiation dependent control of epithelial adherens junction dynamics in physiology, and in EMT and tumor development.

上皮钙粘附素(E-cadherin)介导上皮细胞黏附，并通过β-连环蛋白/α-连环蛋白复合体与肌动蛋白细胞骨架连接，进而促进黏附连接的组织和动力学。上皮细胞表现出器官特有的表型，这种表型在很大程度上是由贴壁连接决定的。我们推测，黏附连接的超分子组织对AJ的组织和功能有重要影响。同样，我们怀疑在EMT过程中间充质钙粘附素如VE-钙粘附素和钙粘附素-11的表达改变了AJ的超分子组织，从而导致了癌症细胞连接动力学的改变。最近，我们发现了一种新的内皮细胞连接调节机制，该机制基于肌动蛋白驱动的连接相关间歇性片层脂蛋白(JAR)。监狱驱动VE-钙粘附素的动力学，从而控制黏附功能、细胞迁移和单层完整性。类似地，我们第一次展示了上皮细胞系和人乳腺癌细胞系显示出类似监狱样的结构。此外，AJ的组织和动力学由肌动蛋白结合和钙粘附素调节蛋白以及涉及acto-myosin介导的收缩的信号分子共同控制。在此背景下，我们确定了LIM和SH3蛋白1(LASP1)，一种肌动蛋白结合蛋白，作为上皮细胞黏附连接的组成部分，并发现它在去分化的乳腺癌MDA细胞系以及慢性炎症组织中过表达，在这种情况下，细胞间黏附的变化起着核心作用。此外，我们确定LASP1是β-连接素的新结合伙伴，它定位于上皮细胞黏附连接，有趣的是，它位于监狱样的质膜突起中。从机制上讲，我们认为LASP1是上皮细胞中一种新的黏附连接相关蛋白，它与肌动蛋白和b-连环蛋白的协同作用有助于连接的组织和动力学。为了测试LASP1和异位表达的钙粘附素对黏附连接动力学的影响，我们将研究不同分化的MDCK细胞系和异位表达钙粘附素-11和VE-钙粘附素的人乳腺癌细胞系。荧光标记的黏附连接和细胞骨架蛋白将被表达，并进行基于荧光的生命细胞成像，以定量监测钙粘附素和肌动蛋白的动态。数据将由超分辨率显微镜补充，以钙粘附素和LASP1依赖的方式表征粘附素连接的超分子组织，以及已建立的细胞和分子生物学技术。我们计划使用CHO表达系统来确认特定的相互作用。我们期望建立一个分子机制模型，帮助解释上皮细胞黏附连接动力学在生理学、EMT和肿瘤发展中的分化依赖控制。