Coordination of Rho GTPase activities during cell protrusion.
Coordination of Rho GTPase activities during cell protrusion.
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GTP酶Rac1、RhoA和Cdc42协同作用以控制细胞骨架动力学。最近的生物传感器研究表明，所有三种GTP酶都在迁移细胞的前端被激活，生化证据表明它们可能相互调节：Cdc42可以激活Rac1，Rac1和RhoA相互抑制。然而，他们的时空协调，在秒和单微米尺寸典型的个别突起事件，仍然未知。在这里，我们研究GTdR协调通过同时可视化两个GTdR生物传感器，并使用“计算复用”的方法能够定义在单独的实验中可视化的多个蛋白质活动之间的关系。我们发现RhoA在细胞边缘被激活，与边缘前进同步，而Cdc 42和Rac 1在边缘后2 μm处被激活，延迟40秒。这表明Rac1和RhoA通过空间分离和精确定时拮抗作用，并且RhoA在突出的初始事件中起作用，而Rac1和Cdc42激活与新扩展的突出的加强和稳定有关的通路。
The GTPases Rac1, RhoA and Cdc42 act in concert to control cytoskeleton dynamics. Recent biosensor studies have shown that all three GTPases are activated at the front of migrating cells and biochemical evidence suggests that they may regulate one another: Cdc42 can activate Rac1, and Rac1 and RhoA are mutually inhibitory. However, their spatiotemporal coordination, at the seconds and single micron dimensions typical of individual protrusion events, remains unknown. Here, we examine GTPase coordination both through simultaneous visualization of two GTPase biosensors and using a “computational multiplexing” approach capable of defining the relationships between multiple protein activities visualized in separate experiments. We found that RhoA is activated at the cell edge synchronous with edge advancement, whereas Cdc42 and Rac1 are activated 2 μm behind the edge with a delay of 40 sec. This indicates that Rac1 and RhoA operate antagonistically through spatial separation and precise timing, and that RhoA plays a role in the initial events of protrusion, while Rac1 and Cdc42 activate pathways implicated in reinforcement and stabilization of newly expanded protrusions.