Identification of stretch-induced biotinylation at cadherin junctions

项目摘要 在胚胎发育或伤口愈合过程中，相邻细胞保持接触并集体迁移。 细胞间黏附在细胞集体迁移过程中的作用尚不清楚。由于不断的拉动和 在迁移的相邻细胞之间推动，我们假设机械力调节 细胞-细胞黏附复合体和肌动蛋白细胞骨架的相互作用，因此， 粘合强度。为了识别细胞-细胞连接处的力敏感蛋白质复合体，我们的创新 生化分析结合了原位近端生物素标记和细胞拉伸装置，促进了 形成力敏复合体。通过将α-连环蛋白与混杂的生物素连接酶融合，任何近端 α-连环蛋白的蛋白质将被生物素化。生物素化过程中的力依赖变化是一个迹象。 改变了的蛋白质复合体。我们的初步研究表明，α-连环蛋白和肌球蛋白IIA可能是 以一种依赖于力的方式相互作用。虽然目前的方法适合于候选人的筛选 力敏感蛋白质，将蛋白质组筛选应用于这种方法将是变革性的， 因为蛋白质组筛选将揭示α-连环蛋白相关蛋白的总组成 外力的存在或不存在，是破译机械-机械的分子基础的关键第一步。 转导。然而，当前协议的关键限制是小信元将限制 蛋白质样本的数量。我们这项提议的目标是重新设计和扩大目前的 分离足以用于质谱学的纯化蛋白质的数量并鉴定 α-连环蛋白周围的力敏感复合体。我们将制造基于硅的细胞拉伸室 薄片作为培养细胞的底物。利用这个装置，我们将优化机械刺激( 频率、底物拉伸的大小和持续时间)基于细胞形态，组织 肌动蛋白细胞骨架，以及生物素化的程度。使用新设计的细胞担架和质量 光谱分析，我们将确定α周围的力敏感分子的全面清单- 连环蛋白对于理解机械转导是必不可少的。

Project Summary During embryogenesis or wound healing, neighboring cells maintain contact and migrate collectively, though the roles of cell-cell adhesion during collective cell migration is poorly defined. Due to constant pulling and pushing between migrating neighboring cells, we hypothesize that mechanical forces regulate the interaction between the cell-cell adhesion complex and the actin cytoskeleton, and therefore, the adhesive strength. To identify force-sensitive protein complexes at cell-cell junctions, our innovative biochemical analysis combines in situ proximal biotin labeling with a cell stretch device that promotes the formation of force-sensitive complexes. By fusing α-catenin with a promiscuous biotin ligase, any proximal proteins of α-catenin will be biotinylated. The force-dependent change in the biotinylation profile is an indication of altered protein complexes. Our preliminary study demonstrates that α-catenin and myosin IIA are likely interacting in a force-dependent manner. While the current approach is suited for the candidate screening of force-sensitive proteins, the application of proteomic screening to this approach will be transformative, because the proteomic screening will reveal the total composition of α-catenin associated proteins in the presence or absence of external forces, a critical first step in deciphering the molecular basis of mechano- transduction. However, the key limitation of the current protocol is that the small cell stretch chambers that limit the quantity of protein samples. Our goal of this proposal is to re-design and scale-up the current protocol to isolate the quantity of purified proteins sufficient for mass spectrometry and identify the force-sensitive complex surrounding α-catenin. We will fabricate cell stretch chambers based on a silicon sheet as a substrate to culture cells. Using this device, we will optimize the mechanical stimulation (the frequency, the magnitude and the duration of substrate stretch) based on cell morphology, the organization of the actin cytoskeleton, and the extent of biotinylation. Using the newly designed cell stretcher and mass spectrometry analysis, we will determine a comprehensive list of the force-sensitive molecules surrounding α- catenin that is essential for understanding of mechano-transduction.