Molecular basis of force-sensing by the keratin network

项目摘要 上皮组织的物理完整性是由细胞骨架网络建立和维持的， 细胞与其他邻近细胞和细胞外基质一起进入其环境。特别地，角蛋白 在上皮细胞中普遍表达的中间丝蛋白负责结构性的 上皮组织的完整性和最近出现的集体细胞迁移的驱动程序。然而，与肌动蛋白不同， 对角蛋白力感测的理解仍然非常有限。我们发现角蛋白网络对 通过招募cten，一种已知既作为肿瘤抑制因子又作为肿瘤抑制因子的蛋白质， 以组织特异性方式启动子。新出现的证据表明，更多的蛋白质被招募到 力轴承角蛋白纤维，这表明角蛋白网络可能作为一个关键枢纽， 机械传导这个应用程序的总体前提是，发现基本机制和 基于角蛋白的机械感测功能将有助于理解细胞如何感测力。 我们的目标是确定上皮细胞角蛋白网络周围的力依赖性蛋白相互作用组， 细胞以及角蛋白介导的机械转导如何影响细胞行为。为了解决力敏 蛋白质-蛋白质相互作用，我们将通过原位混杂生物素标记来标记近端蛋白质，而细胞 都受到了生理刺激新确定的候选人将进行测试，以验证他们的部队依赖的共同， 定位与角蛋白丝在活细胞和体外，并分析其作用，转录 调节、细胞完整性维持和集体细胞迁移。我们的方法将揭示 在存在或不存在外力的情况下，角蛋白相关蛋白的列表，并首次解决 角蛋白网络的力依赖性调节及其生理意义。

Project Summary Physical integrity of epithelial tissue is established and maintained by the cytoskeletal network which integrates cells into their environment with other neighboring cells and the extracellular matrix. In particular, keratin intermediate filament proteins ubiquitously expressed in epithelial cells are responsible for the structural integrity of epithelial tissues and recently emerged as a driver of collective cell migration. Yet, unlike actin, understanding of keratin force-sensing is still very limited. We discovered that the keratin network responds to externally applied physical forces by recruiting cten, a protein known to act as both a tumor suppressor and promoter in a tissue-specific manner. Emerging evidence indicates that more proteins are recruited to the force-bearing keratin fibers, suggesting that the keratin network may serve as a critical hub for mechano-transduction. The overall premise of this application is that discovering the basic mechanisms and functions of keratin-based mechano-sensing will contribute to the understanding of how the cell senses forces. Our goal is to determine the force-dependent protein interactome surrounding the keratin network in epithelial cells and how keratin-mediated mechano-transduction impacts cell behavior. To resolve force-sensitive protein-protein interactions, we will inscribe proximal proteins by in situ promiscuous biotin labeling while cells are being physically stimulated. Newly identified candidates will be tested to verify their force-dependent co- localization with keratin filaments in live cells and in vitro, and analyzed for their roles in transcriptional regulation, cell integrity maintenance, and collective cell migration. Our approach will reveal the comprehensive list of keratin-associated proteins in the presence or absence of external forces and, for the first time, resolve the force-dependent regulation of the keratin network and its physiological implications.