A dual role for Rac1 GTPases in the regulation of cell motility
A dual role for Rac1 GTPases in the regulation of cell motility
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DOI：
10.1242/jcs.089680
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发表时间：
2012-01-15
影响因子：
4
通讯作者：
Weber, Igor
中科院分区：
文献类型：
--
作者：
Filic, Vedrana;Marinovic, Maja;Weber, Igor
Rac 蛋白是盘基网柄菌中唯一典型的 Rho 家族 GTP 酶，它们充当肌动蛋白细胞骨架的关键调节因子。为了监测盘基网柄菌细胞中激活的 Rac1 的动态，开发了一种荧光探针，该探针特异性结合 Rac1 的 GTP 结合形式。该探针基于 PAK1 激酶的 GTP 酶结合域 (GBD)，并根据酵母双杂交、GST 下拉和荧光共振能量转移测定进行选择。 PAK1 GBD 定位于迁移细胞的前缘和内吞杯。因此，与其在脊椎动物中的作用类似，活化的 Rac 1 似乎可以控制盘基网柄菌细胞突出区域的肌动蛋白从头聚合。此外，我们发现 IQGAP 相关蛋白 DGAP1 将活性 Rac 1 与肌动蛋白结合蛋白 Cortexillin I 和 Cortexillin II 隔离成四元复合物，定位于迁移细胞的尾部区域。值得注意的是，PAK1 GBD 和 DGAP1 均与 Rac1-GTP 结合，在细胞迁移、吞噬作用和胞质分裂中表现出相互排斥的定位，以及在化学引诱剂刺激后招募到细胞皮层的相反动力学。此外，PAK1 GBD 的皮质定位取决于肌动蛋白细胞骨架的完整性，而 DGAP1 的皮质定位则不然。综上所述，这些结果表明 Rac1 GTPases 在盘基网柄菌属细胞运动和极性的调节中发挥双重作用。
Rac proteins are the only canonical Rho family GTPases in Dictyostelium, where they act as key regulators of the actin cytoskeleton. To monitor the dynamics of activated Rac1 in Dictyostelium cells, a fluorescent probe was developed that specifically binds to the GTP-bound form of Rac1. The probe is based on the GTPase-binding domain (GBD) from PAK1 kinase, and was selected on the basis of yeast two-hybrid, GST pull-down and fluorescence resonance energy transfer assays. The PAK1 GBD localizes to leading edges of migrating cells and to endocytotic cups. Similarly to its role in vertebrates, activated Rac 1 therefore appears to control de novo actin polymerization at protruding regions of the Dictyostelium cell. Additionally, we found that the IQGAP-related protein DGAP1, which sequesters active Rac 1 into a quaternary complex with actin-binding proteins cortexillin I and cortexillin II, localizes to the trailing regions of migrating cells. Notably, PAK1 GBD and DGAP1, which both bind to Rac1-GTP, display mutually exclusive localizations in cell migration, phagocytosis and cytokinesis, and opposite dynamics of recruitment to the cell cortex upon stimulation with chemoattractants. Moreover, cortical localization of the PAK1 GBD depends on the integrity of the actin cytoskeleton, whereas cortical localization of DGAP1 does not. Taken together, these results imply that Rac1 GTPases play a dual role in regulation of cell motility and polarity in Dictyostelium.