Cadherin-catenin regulation in dividing epithelial cells

钙粘附素-连环蛋白细胞-细胞黏附复合体对于组织发育和动态平衡是必不可少的。而当 每种成分对细胞间黏附的要求早已确定，但我们所知的要少得多 关于调控这种复合体的结构和化学修饰。理解的一个障碍 钙粘附素/连环蛋白的黏附调控缺乏一个明确的系统来分析。第二个障碍是 连环蛋白在钙粘附素-连环蛋白复合体之外发挥功能，混淆了功能丧失的分析。单元格 在上皮内的分裂经历了刻板的形状变化来分离基因组，同时也 维持粘附性屏障，但如何调节钙粘附素-连环蛋白复合体以适应 这一基本过程是未知的。我们已经产生了一种α-连环蛋白的变体，它显示了增强的结合F- 肌动蛋白在体外。用该变体恢复的由CRISPR/Cas9生成的α-连环蛋白敲除细胞显示出显著的 有丝分裂缺陷，尤指在胞质分裂期间。这表明α-连环蛋白与F-之间的持续偶联。 肌动蛋白阻断细胞分裂，提示α-连环蛋白/F-肌动蛋白相互作用可能在有丝分裂过程中受到调节 (目标1)。我们已经在α-连环蛋白中发现了一种进化上保守的磷酸化方案，它位于 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，每个连接区都是 对于哺乳动物细胞和果蝇的α-连环蛋白黏附功能是必不可少的。当这种磷酸化被上调时 在有丝分裂过程中，我们假设α-连环蛋白的磷酸化通过改变肌动蛋白来调节细胞分裂。 α-连环蛋白的结合和/或机械传感特性(目标2)。最后，我们发现一种形式的α-连锁素 钙粘蛋白复合体外的功能可以结合富含磷脂酰肌醇-3，4，5-三磷酸(PIP3) 膜和可能与有丝分裂相关的直接皮质肌动蛋白组织(目标3)。总而言之， 这项提议融合了多学科合作调查团队的技能，使用细胞系， 果蝇，机械和生化方法，以解决α-连环蛋白是如何形成这一基本问题的 有丝分裂过程中的结构改变，这将对不同物种之间的黏附调节产生广泛的影响 单元类型。

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.