Cadherin-catenin regulation in dividing epithelial cells

钙粘蛋白-连环蛋白细胞-细胞粘附复合物对于组织发育和稳态是必不可少的。而 细胞间粘附对每种成分要求早已确定，但我们知道的却少得多 关于调节这种复合物的结构和化学修饰。理解的一个障碍是 钙粘蛋白/连环蛋白粘附调节缺乏明确的分析系统。第二个障碍是， 连环蛋白在钙粘蛋白-连环蛋白复合物之外发挥功能，混淆了功能丧失分析。细胞 上皮内的分裂经历刻板的形状变化以分离基因组， 维持粘附屏障，但如何调节钙粘蛋白-连环蛋白复合体以适应 这个基本过程是未知的。我们已经产生了一种α-连环蛋白的变体，它显示出增强的F- 体外肌动蛋白。用这种变体恢复的CRISPR/Cas9产生的α-连环蛋白敲除细胞显示出显著的细胞毒性。 有丝分裂缺陷，特别是在胞质分裂期间。这表明α-连环蛋白和F- 肌动蛋白阻断细胞分裂，表明α-catenin/F-actin相互作用可能在有丝分裂过程中受到调节 (Aim 1）。我们已经确定了α-连环蛋白中进化上保守的磷酸化机制， 连接α-连环蛋白的肌动蛋白结合区和机械敏感区的连接区，每个区域都是 在哺乳动物细胞和果蝇中对α-连环蛋白粘附功能至关重要。随着磷酸化水平的上调 在有丝分裂过程中，我们假设α-连环蛋白磷酸化通过改变肌动蛋白- α-连环蛋白的结合和/或机械传感特性（目的2）。最后，我们发现一种α-连环蛋白， 钙粘蛋白复合物外的功能可以结合富含磷脂酰肌醇-3，4，5-三磷酸（PIP 3）的 膜和直接皮质肌动蛋白组织，可能与有丝分裂有关（目的3）。总之， 该提案结合了来自多学科合作研究者团队的技能，使用细胞系， 果蝇，机械和生物化学方法来解决α-连环蛋白是如何 在有丝分裂过程中进行结构修饰，这将对多种细胞的粘附调节产生广泛的影响。 细胞类型。

The cadherin-catenin cell-cell adhesion complex is essential for tissue development and homeostasis. While the requirement of each component to intercellular adhesion has been long established, we know much less about the structural and chemical modifications that regulate this complex. One barrier to understanding cadherin/catenin adhesion regulation is the lack of a well-defined system for analysis. A second barrier is that catenins serve functions outside of the cadherin-catenin complex, confounding loss of function analyses. Cells dividing within an epithelium undergo stereotypic shape changes to separate the genome while also maintaining the adhesive barrier, but how the cadherin-catenin complex might be regulated to accommodate this essential process is unknown. We have generated a variant of α-catenin that shows enhanced binding F- actin in vitro. CRISPR/Cas9-generated α-catenin knockout cells restored with this variant show a prominent mitotic defect, particularly during cytokinesis. This shows that persistent coupling between α-catenin and F- actin blocks cell division, and suggests that the α-catenin/F-actin interaction may be regulated during mitosis (Aim 1). We've identified an evolutionarily conserved phosphorylation scheme in α-catenin that lies within a linker region that joins the actin-binding and mechano-sensitive regions of α-catenin, each of which are essential for α-catenin adhesive function in mammalian cells and flies. As this phosphorylation is upregulated during mitosis, we hypothesize that α-catenin phosphorylation regulates cell division by altering the actin- binding and/or mechanosensing property of α-catenin (Aim 2). Lastly, we've found that a form of α-catenin that functions outside of the cadherin complex can bind phosphatidylinositol-3,4,5-trisphosphate (PIP3)-enriched membranes and direct cortical actin organizations that might be relevant to mitotic division (Aim 3). Altogether, this proposal incorporates skills from a multi-disciplinary team of collaborative investigators, using cell lines, Drosophila, mechanical and biochemical approaches to address the fundamental question of how α-catenin is structurally modified during mitosis, which will have broad implications for adhesion regulation across diverse cell types.