Department of Biomedical Engineering, Duke University, Durham, North Carolina.
Lineberger Comprehensive Cancer Center, UNC Chapel, Chapel Hill, North Carolina.
Biophys J. 2018 Apr 10;114(7):1680-1694. doi: 10.1016/j.bpj.2018.02.019.
Cell migration is a complex process, requiring coordination of many subcellular processes including membrane protrusion, adhesion, and contractility. For efficient cell migration, cells must concurrently control both transmission of large forces through adhesion structures and translocation of the cell body via adhesion turnover. Although mechanical regulation of protein dynamics has been proposed to play a major role in force transmission during cell migration, the key proteins and their exact roles are not completely understood. Vinculin is an adhesion protein that mediates force-sensitive processes, such as adhesion assembly under cytoskeletal load. Here, we elucidate the mechanical regulation of vinculin dynamics. Specifically, we paired measurements of vinculin loads using a Förster resonance energy transfer-based tension sensor and vinculin dynamics using fluorescence recovery after photobleaching to measure force-sensitive protein dynamics in living cells. We find that vinculin adopts a variety of mechanical states at adhesions, and the relationship between vinculin load and vinculin dynamics can be altered by the inhibition of vinculin binding to talin or actin or reduction of cytoskeletal contractility. Furthermore, the force-stabilized state of vinculin required for the stabilization of membrane protrusions is unnecessary for random migration, but is required for directional migration along a substrate-bound cue. These data show that the force-sensitive dynamics of vinculin impact force transmission and enable the mechanical integration of subcellular processes. These results suggest that the regulation of force-sensitive protein dynamics may have an underappreciated role in many cellular processes.
细胞迁移是一个复杂的过程，需要协调许多亚细胞过程，包括膜突出、粘附和收缩。为了实现有效的细胞迁移，细胞必须同时控制通过粘附结构传递的大力量和通过粘附周转率的细胞体的易位。虽然已经提出了蛋白质动力学的机械调节在细胞迁移过程中的力传递中起主要作用，但关键蛋白质及其确切作用尚未完全理解。粘着斑蛋白是一种粘附蛋白，介导力敏感过程，例如在细胞骨架负载下的粘附组装。在这里，我们阐明了粘着斑蛋白动力学的机械调节。具体来说，我们通过基于Förster 共振能量转移的张力传感器测量粘着斑蛋白的张力，并通过光漂白后荧光恢复测量粘着斑蛋白动力学，以测量活细胞中力敏感蛋白动力学。我们发现粘着斑蛋白在黏附处采用多种机械状态，并且粘着斑蛋白的负载和粘着斑蛋白动力学之间的关系可以通过抑制粘着斑蛋白与钙调蛋白或肌动蛋白的结合或降低细胞骨架收缩性来改变。此外，稳定膜突出所必需的粘着斑蛋白的力稳定状态对于随机迁移不是必需的，但对于沿着基底结合的线索进行定向迁移是必需的。这些数据表明粘着斑蛋白的力敏感动力学影响力传递，并能够实现亚细胞过程的机械整合。这些结果表明，力敏感蛋白动力学的调节可能在许多细胞过程中具有被低估的作用。
