Identification of stretch-induced biotinylation at cadherin junctions

钙粘蛋白连接处拉伸诱导的生物素化的鉴定

• 批准号: 9182555
• 负责人: Soichiro Yamada
• 金额: $ 7.08万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9182555
• 关键词: Actins; Adhesions; Adhesives; Area; Biochemical; Biochemistry; Biotin; Biotinylation; Cadherins; Cell Communication; Cell Count; Cell-Cell Adhesion; Cells; Cellular Morphology; Complex; Cultured Cells; Custom; Cytoskeleton; Data; Detection; Devices; Embryonic Development; Frequencies; Goals; Home environment; In Situ; In Vitro; Individual; Intercellular Junctions; Label; Lavandula; Length; Ligase; Malignant Epithelial Cell; Mass Spectrum Analysis; Mechanical Stimulation; Mechanics; Membrane; Molecular; Motor; Neoplasm Metastasis; Nonmuscle Myosin Type IIA; Organ; Performance; Phenotype; Positioning Attribute; Precipitation; Property; Proteins; Proteomics; Protocols documentation; Regulation; Role; Sampling; Silicon; Silver Staining; Slide; Staging; Streptavidin; Stretching; Techniques; Tissues; Variant; Western Blotting; Wound Healing; alpha catenin; base; cell motility; design; flexibility; innovation; new therapeutic target; programs; protein complex; protein profiling; protein protein interaction; prototype; scale up; screening

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.

项目摘要 在胚胎发生或伤口愈合过程中，相邻细胞保持接触并集体迁移， 在集体细胞迁移过程中细胞-细胞粘附的作用还不清楚。由于不断的拉扯， 在迁移的相邻细胞之间推动，我们假设机械力调节细胞的运动。 细胞-细胞粘附复合物和肌动蛋白细胞骨架之间的相互作用，因此， 粘合强度为了识别细胞-细胞连接处的力敏感蛋白复合物，我们的创新 生物化学分析将原位近端生物素标记与细胞拉伸装置相结合， 力敏感复合物的形成。通过将α-连环蛋白与混杂的生物素连接酶融合， α-连环蛋白的蛋白质将被生物素化。生物素化谱的力依赖性变化是一个迹象 改变的蛋白质复合物。我们的初步研究表明，α-连环蛋白和肌球蛋白IIA可能是 以依赖于力的方式相互作用。虽然目前的办法适用于候选人筛选， 力敏感蛋白，蛋白质组学筛选应用于这种方法将是变革性的， 因为蛋白质组学筛选将揭示α-连环蛋白相关蛋白的总组成， 存在或不存在外力，这是破译机械的分子基础的关键的第一步， 转导然而，当前方案的关键限制是限制细胞生长的小细胞拉伸室。 蛋白质样品的数量。我们提出这项建议的目的是重新设计和扩大目前的 分离足以用于质谱分析的纯化蛋白质的量并鉴定 围绕α-连环蛋白的力敏复合物。我们将制造基于硅的细胞拉伸室 片作为培养细胞基质。使用该设备，我们将优化机械刺激（ 频率，幅度和持续时间的基板拉伸）的基础上细胞形态，组织 肌动蛋白细胞骨架和生物素化的程度。使用新设计的细胞拉伸器和质量 光谱分析，我们将确定围绕α- 连环蛋白是理解机械传导所必需的。