Mechanical forces alter zyxin unbinding kinetics within focal adhesions of living cells
Mechanical forces alter zyxin unbinding kinetics within focal adhesions of living cells
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DOI：
10.1002/jcp.20550
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发表时间：
2006-04-01
影响因子：
5.6
通讯作者：
Ingber, DE
中科院分区：
文献类型：
--
作者：
Lele, TP;Pendse, J;Ingber, DE
介导细胞形状和运动改变的局灶粘连的形成是由一种机械化学机制控制的，其中细胞骨架张力驱动分子组装的变化；然而，对这种反应的分子生物物理基础知之甚少。在这里，我们描述了一种通过修改光漂白后荧光恢复（FRAP）技术并将其与数学建模相结合来测量活细胞中单个gfp标记的焦点粘附分子的解结合速率常数k（OFF）的方法。通过这种方法，我们发现通过三种不同的技术——化学抑制细胞骨架张力的产生，激光切割相关的肌动蛋白应力纤维，或使用柔顺的细胞外基质——可以降低细胞对黏着蛋白酶的牵引力，从而增加黏着蛋白酶的k（OFF）。相比之下，另一种黏附蛋白vinculin的k（OFF）在张力消散后保持不变。数学模型也表明，这些发生在数秒内的力依赖的增效酶k（OFF）的增加足以定量预测在生理上发生在数分钟内的大规模局灶黏附分解。这些发现表明，一些（但不是全部）局灶黏附蛋白的分子结合动力学对机械力敏感，并表明这种生物物理参数的力依赖性变化可能控制着活细胞中局灶黏附重塑背后的超分子事件。
The formation of focal adhesions that mediate alterations of cell shape and movement is controlled by a mechanochemical mechanism in which cytoskeletal tensional forces drive changes in molecular assembly; however, little is known about the molecular biophysical basis of this response. Here, we describe a method to measure the unbinding rate constant k(OFF) of individual GFP-labeled focal adhesion molecules in living cells by modifying the fluorescence recovery after photobleaching (FRAP) technique and combining it with mathematical modeling. Using this method, we show that decreasing cellular traction forces on focal adhesions by three different techniques-chemical inhibition of cytoskeletal tension generation, laser incision of an associated actin stress fiber, or use of compliant extracellular matrices-increases the k(OFF) of the focal adhesion protein zyxin. In contrast, the k(OFF) of another adhesion protein, vinculin, remains unchanged after tension dissipation. Mathematical models also demonstrate that these force-dependent increases in zyxin's k(OFF) that Occur over seconds are sufficient to quantitatively predict large-scale focal adhesion disassembly that occurs physiologically over many minutes. These findings demonstrate that the molecular binding kinetics of some, but not all, focal adhesion proteins are sensitive to mechanical force, and suggest that force-dependent changes in this biophysical parameter may govern the supramolecular events that underlie focal adhesion remodeling in living cells.