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

上皮钙粘蛋白（E-cadherin）介导上皮粘附并通过β-连环蛋白/α-连环蛋白复合物连接到肌动蛋白细胞骨架，肌动蛋白细胞骨架反过来有助于粘附连接组织和动力学。上皮细胞表现出一种器官特异性的表型，这种表型在很大程度上是由粘附连接决定的。我们推测粘附连接的超分子组织对AJs的组织和功能有显著影响。同样，我们怀疑EMT期间间充质钙粘蛋白（如VE-钙粘蛋白和钙粘蛋白-11）的表达改变了AJs的超分子组织，从而导致癌细胞连接动力学的改变。近年来，我们发现了一种新的内皮细胞连接调节机制，即肌动蛋白驱动的连接相关间歇性板状伪足（JAIL）。JAIL驱动VE-钙粘蛋白动力学，从而控制粘附特征、细胞迁移和单层完整性。同样，在这里，我们第一次表明，上皮细胞系和人乳腺癌细胞系显示可比较的JAIL样结构。此外，AJ的组织和动力学是由肌动蛋白结合和钙粘蛋白调节蛋白以及涉及肌动蛋白介导的收缩性的信号分子控制的。在这种情况下，我们确定了LIM和SH 3蛋白1（LASP 1），肌动蛋白结合蛋白，作为上皮粘附连接的组成部分，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，细胞间粘附的改变起着核心作用。此外，我们确定LASP 1作为β-catenin的新结合伴侣，其定位于上皮粘附连接处，并且有趣地定位于JAIL样质膜突起。从机制上讲，我们提出LASP 1作为一种新的粘附连接相关的蛋白在上皮细胞，在协同相互作用与肌动蛋白和β-连环蛋白有助于连接组织和动力学。为了测试LASP 1和异位表达的钙粘蛋白对粘附连接动力学的影响，我们将研究异位表达钙粘蛋白-11和VE-钙粘蛋白的不同分化的MDCK细胞系和人乳腺癌细胞系。将表达荧光标记的粘附连接和细胞骨架蛋白，并进行基于荧光的生命细胞成像以定量监测钙粘蛋白和肌动蛋白动力学。数据将通过超分辨率显微镜进行补充，以钙粘蛋白和LASP 1依赖性方式表征粘附连接的超分子组织，沿着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。我们希望建立一个分子机制模型，有助于解释分化依赖性控制的上皮粘附连接动力学在生理学，EMT和肿瘤的发展。

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.