Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA; School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, USA.
Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA; Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA; Department of Chemistry, Queen Mary University of London, London, UK.
Biomaterials. 2024 Jul;308:122542. doi: 10.1016/j.biomaterials.2024.122542. Epub 2024 Mar 20.
Focal adhesions (FAs) are nanoscale complexes containing clustered integrin receptors and intracellular structural and signaling proteins that function as principal sites of mechanotransduction in part via promoting the nuclear translocation and activation of the transcriptional coactivator yes-associated protein (YAP). Knockdown of FA proteins such as focal adhesion kinase (FAK), talin, and vinculin can prevent YAP nuclear localization. However, the mechanism(s) of action remain poorly understood. Herein, we investigated the role of different functional domains in vinculin, talin, and FAK in regulating YAP nuclear localization. Using genetic or pharmacological inhibition of fibroblasts and human mesenchymal stem cells (hMSCs) adhering to deformable substrates, we find that disruption of vinculin-talin binding versus talin-FAK binding reduces YAP nuclear localization and transcriptional activity via different mechanisms. Disruption of vinculin-talin binding or knockdown of talin-1 reduces nuclear size, traction forces, and YAP nuclear localization. In contrast, disruption of the talin binding site on FAK or elimination of FAK catalytic activity did not alter nuclear size yet still prevented YAP nuclear localization and activity. These data support both nuclear tension-dependent and independent models for matrix stiffness-regulated YAP nuclear localization. Our results highlight the importance of vinculin-talin-FAK interactions at FAs of adherent cells, controlling YAP nuclear localization and activity.
焦点黏附（FAs）是纳米级复合物，包含聚集的整合素受体和细胞内结构和信号蛋白，通过促进核转位和转录共激活因子 yes 相关蛋白（YAP）的激活，在机械转导中起主要作用。FA 蛋白（如黏着斑激酶（FAK）、桩蛋白和 vinculin）的敲低可阻止 YAP 核定位。然而，其作用机制仍知之甚少。在此，我们研究了 vinculin、talin 和 FAK 中的不同功能域在调节 YAP 核定位中的作用。通过对黏附于可变形底物的成纤维细胞和人间充质干细胞（hMSCs）进行遗传或药理学抑制，我们发现破坏 vinculin-talin 结合与 talin-FAK 结合会通过不同的机制减少 YAP 的核定位和转录活性。破坏 vinculin-talin 结合或敲低 talin-1 会减小核大小、牵引力和 YAP 核定位。相比之下，破坏 FAK 上的 talin 结合位点或消除 FAK 催化活性不会改变核大小，但仍可阻止 YAP 核定位和活性。这些数据支持了基质刚度调节 YAP 核定位的核张力依赖性和非依赖性模型。我们的结果强调了黏着斑处 vinculin-talin-FAK 相互作用的重要性，控制着 YAP 的核定位和活性。
