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，该网络整合了 细胞与其他邻近细胞和细胞外基质一起进入它们的环境。尤其是角蛋白 在上皮细胞中普遍表达的中间丝蛋白负责结构 上皮组织的完整性，最近成为集体细胞迁移的驱动力。然而，与肌动蛋白不同， 对角蛋白的力觉知觉的了解仍然非常有限。我们发现角蛋白网络对 通过招募cten来施加外部物理力量，cten是一种已知的既是肿瘤抑制因子又是 启动子以组织特异性的方式。新出现的证据表明，更多的蛋白质被招募到 承载角蛋白的纤维，提示角蛋白网络可能是 机械转导。这个应用程序的总体前提是发现基本机制和 基于角蛋白的机械传感功能将有助于理解细胞如何感知力。 我们的目标是确定上皮细胞角蛋白网络周围的力依赖蛋白相互作用体 以及角蛋白介导的机械转导如何影响细胞行为。解析力敏感 蛋白质之间的相互作用，我们将通过原位杂交性生物素标记来标记近端蛋白质，而细胞 都被身体刺激了。新确定的候选人将接受测试，以验证他们依赖武力的共同 角蛋白细丝在活细胞和体外的定位，并分析它们在转录中的作用 调节、细胞完整性维护和集体细胞迁移。我们的方法将揭示全面的 角蛋白相关蛋白在外力存在或不存在的情况下的列表，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.