FRET binding antenna reports spatiotemporal dynamics of GDI-Cdc42 GTPase interactions.
FRET binding antenna reports spatiotemporal dynamics of GDI-Cdc42 GTPase interactions.
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DOI：
10.1038/nchembio.2145
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发表时间：
2016-10
影响因子：
14.8
通讯作者：
Hahn KM
中科院分区：
文献类型：
--
作者：
Hodgson L;Spiering D;Sabouri-Ghomi M;Dagliyan O;DerMardirossian C;Danuser G;Hahn KM
鸟嘌呤-核苷酸解离抑制剂（GDI）是Rho家族GTP酶的负调节剂，其将GTP酶与膜隔离。在这里，我们问GDI-Cdc 42相互作用如何调节局部Cdc 42激活细胞运动。细胞对Rho家族途径组分过表达的敏感性使我们产生了一种新的生物传感器设计（GDI.Cdc42 FLARE），其中Cdc 42用选择性报告Cdc 42与内源性GDI结合的FRET“结合天线”修饰。类似的触角也可以报告GDI-Rac 1和GDI-RhoA相互作用。通过计算多路复用和同步成像，我们确定了GDI-Cdc 42相互作用和Cdc 42在细胞突起和收缩过程中的激活的时空动力学。这揭示了在10秒的时间尺度上GTdR释放和激活的显著紧密协调，表明GDI-Cdc 42相互作用是Cdc 42活性的时空调节中的关键组分，而不仅仅是用于全局螯合失活的信号分子池的机制。
Guanine-nucleotide dissociation inhibitors (GDI) are negative regulators of Rho family GTPases that sequester the GTPases away from the membrane. Here we ask how GDI-Cdc42 interaction regulates localized Cdc42 activation for cell motility. The sensitivity of cells to overexpression of Rho family pathway components led us to a new biosensor design (GDI.Cdc42 FLARE), in which Cdc42 was modified with a FRET ‘binding antenna’ that selectively reported Cdc42 binding to endogenous GDI. Similar antennae could also report GDI-Rac1 and GDI-RhoA interaction. Through computational multiplexing and simultaneous imaging, we determined the spatiotemporal dynamics of GDI-Cdc42 interaction and Cdc42 activation during cell protrusion and retraction. This revealed a remarkably tight coordination of GTPase release and activation on a time scale of 10 seconds, suggesting that GDI-Cdc42 interactions are a critical component in the spatiotemporal regulation of Cdc42 activity, and not merely a mechanism for global sequestration of an inactivated pool of signaling molecules.