Department of Physics and Astronomy, Iowa State University, Ames, Iowa.
Department of Physics and Astronomy, Iowa State University, Ames, Iowa; Department of Biochemistry, Biophysics and Molecular Biology, Ames, Iowa.
Biophys J. 2023 Jan 3;122(1):156-167. doi: 10.1016/j.bpj.2022.11.013. Epub 2022 Nov 9.
Focal adhesions (FAs) transmit force and mediate mechanotransduction between cells and the matrix. Previous studies revealed that integrin-transmitted force is critical to regulate FA formation. As vinculin is a prominent FA protein implicated in integrin tension transmission, this work studies the relation among integrin tensions (force), vinculin (protein), and FA formation (structure) by integrin tension manipulation, force visualization and vinculin knockout (KO). Two DNA-based integrin tension tools are adopted: tension gauge tether (TGT) and integrative tension sensor (ITS), with TGT restricting integrin tensions under a designed T (tension tolerance) value and ITS visualizing integrin tensions above the T value by fluorescence. Results show that large FAs (area >1 μm) were formed on the TGT surface with T of 54 pN but not on those with lower T values. Time-series analysis of FA formation shows that focal complexes (area <0.5 μm) appeared on all TGT surfaces 20 min after cell plating, but only matured to large FAs on TGT with T of 54 pN. Next, we tested FA formation in vinculin KO cells on TGT surfaces. Surprisingly, the T value of TGT required for large FA formation is drastically decreased to 23 pN. To explore the cause, we visualized integrin tensions in both wild-type and vinculin KO cells using ITS. The results showed that integrin tensions in FAs of wild-type cells frequently activate ITS with T of 54 pN. With vinculin KO, however, integrin tensions in FAs became lower and unable to activate 54 pN ITS. Force signal intensities of integrin tensions reported by 33 and 43 pN ITS were also significantly reduced with vinculin KO, suggesting that vinculin is essential to transmit high-level integrin tensions and involved in transmitting intermediate-level integrin tensions in FAs. However, the high-level integrin tensions transmitted by vinculin are not required by FA formation.
黏着斑（FAs）在细胞和基质之间传递力并介导机械转导。先前的研究表明，整合素传递的力对于调节 FA 形成至关重要。由于 vinculin 是一种在整合素张力传递中起重要作用的 FA 蛋白，本工作通过整合素张力操作、力可视化和 vinculin 敲除（KO）研究了整合素张力（力）、vinculin（蛋白）和 FA 形成（结构）之间的关系。采用了两种基于 DNA 的整合素张力工具：张力计系绳（TGT）和整合张力传感器（ITS），其中 TGT 在设计的 T（张力容忍度）值下限制整合素张力，而 ITS 通过荧光可视化 T 值以上的整合素张力。结果表明，在 TGT 表面形成了具有 54 pN 的 T 值的大 FA（面积>1 μm），而在 T 值较低的表面则没有形成。FA 形成的时间序列分析表明，在细胞接种后 20 分钟，所有 TGT 表面上均出现了焦点复合物（面积<0.5 μm），但仅在 T 值为 54 pN 的 TGT 上成熟为大 FA。接下来，我们在 TGT 表面上测试了 vinculin KO 细胞的 FA 形成。令人惊讶的是，用于大 FA 形成的 TGT 的 T 值急剧降低至 23 pN。为了探究原因，我们使用 ITS 在野生型和 vinculin KO 细胞中可视化整合素张力。结果表明，野生型细胞 FA 中的整合素张力经常激活 T 值为 54 pN 的 ITS。然而，由于 vinculin KO，FA 中的整合素张力降低，无法激活 54 pN ITS。vinculin KO 后，报告的 33 和 43 pN ITS 的整合素张力的力信号强度也明显降低，这表明 vinculin 对于传递高水平的整合素张力以及参与 FA 中的中间水平的整合素张力传递是必不可少的。然而，FA 形成不需要由 vinculin 传递的高水平整合素张力。
