Formin-like 2 associates with the alpha-catenin/E-cadherin complex to control junctional actin dynamics and epithelialization

单个上皮细胞之间粘附连接的新生形成需要协调的空间肌动蛋白动力学来产生专门的局部力。调控细胞-细胞粘附起始和维持的新生肌动蛋白聚合的机制尚不清楚。在这里，我们在三维环境中研究了人乳腺上皮细胞系MCF10A在子细胞-细胞粘附形成过程中形成蛋白的组装。我们发现，在新形成的细胞-细胞接触中，肌动蛋白的组装和周转以及新生粘附连接的形成都需要FMNL2。在长期3D培养中，FMNL2的抑制导致上皮管腔形成严重破坏。我们发现FMNL2以一种受调控的方式定位于细胞-细胞接触，FMNL2与α -catenin和E-Cadherin形成复合物，这种复合物在Rac1活性的强烈促进下形成。光遗传学控制的LOV-Rac1的光激活迅速驱动FMNL2到连接区进行局部肌动蛋白组装，而光漂白实验表明，FMNL2弥补了Rac1在连接肌动蛋白转换上的缺陷。因此，FMNL2似乎是人类3D上皮培养中新生连接肌动蛋白组装和从头粘附连接形成的关键因素。因此，这些发现需要详细了解formmin诱导的肌动蛋白聚合和细胞-细胞粘附之间的机制关系。本提案的目的是深入研究FMNL2如何整合到AJ复合体中，以及这种相互作用是否参与FMNL2的调控，结合3D细胞培养，生物化学，光遗传学工具的发展以及活细胞成像技术。这些研究将对形成蛋白在完整的人上皮细胞内细胞接触中的作用提供更深入的了解。

De novo formation of adherens junctions between single epithelial cells requires coordinated, spatial actin dynamics for specialized local force generation. The mechanisms steering nascent actin polymerization for cell-cell adhesion initiation and maintenance are only poorly understood. Here we investigate actin assembly by formins during daughter cell-cell adhesion formation in the human breast epithelial cell line MCF10A in a 3D environment. We identify formin-like 2 (FMNL2) to be required for actin assembly and turnover at newly formed cell-cell contacts as well as for de novo adherens junction formation. In long-term 3D cultures, the suppression of FMNL2 results in severely disrupted epithelial lumen formation. We find that FMNL2 localizes to cell-cell contacts in a regulated manner and FMNL2 forms a complex with alpha-catenin and E-Cadherin, which is strongly promoted by Rac1 activity. Photoactivation of an optogenetically controlled LOV-Rac1 rapidly drives FMNL2 to the junctional zone for localized actin assembly, while photobleaching experiments reveal that FMNL2 rescues Rac1 deficiency on junctional actin turnover. Thus, FMNL2 appears to be a critical factor for nascent junctional actin assembly and de novo adheres junction formation in human 3D epithelial cultures. Consequently, these findings require a detailed understanding of the mechanistic relationship between formin-induced actin polymerization and cell-cell adhesion. The aim of this proposal is to examine in depth, how FMNL2 is integrated into the AJ complex and whether this interaction participates in the regulation of FMNL2 using a combination of 3D cell culture, biochemistry, the development of optogenetic tools as well as live cell imaging techniques. These studies shall provide a deeper understanding on the roles of formins at cell-cell contacts within an intact human epithelium.