A genetically encoded photoactivatable Rac controls the motility of living cells.
A genetically encoded photoactivatable Rac controls the motility of living cells.
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蛋白质活性的精确时空动力学通常在确定细胞行为方面至关重要，但对于大多数蛋白质，它们仍然知之甚少;仍然难以在活细胞内的精确时间和地点操纵蛋白质活性。蛋白质活性已被控制的光，通过蛋白质衍生与光裂解部分或使用光反应性小分子配体。然而，这需要使用有毒的UV波长，活化是不可逆的，和/或通过破坏细胞膜（即通过显微注射）完成细胞加载。我们已经开发出一种新的方法来生产基因编码的光活化衍生物的Rac 1，一个关键的GTdR调节肌动蛋白细胞骨架动力学。将Rac 1突变体融合到来自phototropin的光反应性LOV（光氧电压）结构域，空间上阻断Rac 1相互作用，直到照射解开连接LOV与Rac 1的螺旋。光活化Rac 1（PA-Rac 1）可以使用458或473 nm的光可逆地和重复地激活，以产生精确定位的细胞突起和皱褶。局部Rac激活或失活足以产生细胞运动性并控制细胞运动的方向。肌球蛋白参与Rac控制方向性，但不参与Rac诱导的突出，而PAK是Rac诱导的突出所必需的。PA-Rac 1用于阐明RhoA在细胞运动中的Rac调节。Rac和Rho以秒和亚微米的精度协调细胞骨架行为。它们的相互调节仍然存在争议，数据表明Rac抑制和/或激活Rho。Rac显示抑制活细胞中的RhoA，在突起和皱褶处调节抑制。PA-Rac晶体结构和建模揭示了LOV-Rac相互作用，这将有助于将这种光活化方法扩展到其他蛋白质。
The precise spatio-temporal dynamics of protein activity are often critical in determining cell behaviour, yet for most proteins they remain poorly understood; it remains difficult to manipulate protein activity at precise times and places within living cells. Protein activity has been controlled by light, through protein derivatization with photocleavable moieties or using photoreactive small molecule ligands. However, this requires use of toxic UV wavelengths, activation is irreversible, and/or cell loading is accomplished via disruption of the cell membrane (i.e. through microinjection). We have developed a new approach to produce genetically-encoded photo-activatable derivatives of Rac1, a key GTPase regulating actin cytoskeletal dynamics. Rac1 mutants were fused to the photoreactive LOV (light oxygen voltage) domain from phototropin, sterically blocking Rac1 interactions until irradiation unwound a helix linking LOV to Rac1. Photoactivatable Rac1 (PA-Rac1) could be reversibly and repeatedly activated using 458 or 473 nm light to generate precisely localized cell protrusions and ruffling. Localized Rac activation or inactivation was sufficient to produce cell motility and control the direction of cell movement. Myosin was involved in Rac control of directionality but not in Rac-induced protrusion, while PAK was required for Rac-induced protrusion. PA-Rac1 was used to elucidate Rac regulation of RhoA in cell motility. Rac and Rho coordinate cytoskeletal behaviours with seconds and submicron precision. Their mutual regulation remains controversial, with data indicating that Rac inhibits and/or activates Rho. Rac was shown to inhibit RhoA in living cells, with inhibition modulated at protrusions and ruffles. A PA-Rac crystal structure and modelling revealed LOV-Rac interactions that will facilitate extension of this photoactivation approach to other proteins.