van Unen Jakobus, Reinhard Nathalie R, Yin Taofei, Wu Yi I, Postma Marten, Gadella Theodorus W J, Goedhart Joachim
Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, P.O. Box 94215, NL-1090 GE Amsterdam, The Netherlands.
Center for Cell Analysis and Modeling, University of Connecticut Health Center, 400 Farmington Avenue, Farmington, CT 06032-6406.
Sci Rep. 2015 Oct 5;5:14693. doi: 10.1038/srep14693.
The small GTPase RhoA is involved in cell morphology and migration. RhoA activity is tightly regulated in time and space and depends on guanine exchange factors (GEFs). However, the kinetics and subcellular localization of GEF activity towards RhoA are poorly defined. To study the mechanism underlying the spatiotemporal control of RhoA activity by GEFs, we performed single cell imaging with an improved FRET sensor reporting on the nucleotide loading state of RhoA. By employing the FRET sensor we show that a plasma membrane located RhoGEF, p63RhoGEF, can rapidly activate RhoA through endogenous GPCRs and that localized RhoA activity at the cell periphery correlates with actin polymerization. Moreover, synthetic recruitment of the catalytic domain derived from p63RhoGEF to the plasma membrane, but not to the Golgi apparatus, is sufficient to activate RhoA. The synthetic system enables local activation of endogenous RhoA and effectively induces actin polymerization and changes in cellular morphology. Together, our data demonstrate that GEF activity at the plasma membrane is sufficient for actin polymerization via local RhoA signaling.
小GTP酶RhoA参与细胞形态和迁移。RhoA活性在时间和空间上受到严格调控，且依赖于鸟嘌呤交换因子（GEFs）。然而，GEF对RhoA的活性动力学和亚细胞定位尚不清楚。为了研究GEFs对RhoA活性进行时空控制的潜在机制，我们使用了一种改进的FRET传感器对RhoA的核苷酸负载状态进行单细胞成像。通过使用该FRET传感器，我们发现位于质膜的RhoGEF，即p63RhoGEF，可通过内源性GPCR快速激活RhoA，并且细胞周边局部的RhoA活性与肌动蛋白聚合相关。此外，将源自p63RhoGEF的催化结构域人工募集至质膜而非高尔基体，足以激活RhoA。该合成系统能够局部激活内源性RhoA，并有效诱导肌动蛋白聚合和细胞形态变化。总之，我们的数据表明质膜处的GEF活性足以通过局部RhoA信号传导实现肌动蛋白聚合。
