监测活细胞中诱导激活的Rac、Cdc42信号转导通路的FRET单分子探针

FRET-BASED SINGLE-MOLECULE PROBES FOR MONITORING INDUCED ACTIVATION OF RAC,CDC42 SIGNALING PATHWAYS IN LIVING CELLS

Rho家族鸟苷三磷酸酶,包括Rac1和Cdc42等,参与调节细胞形态、细胞迁移、转录激活和基因表达等一系列细胞过程。根据FRET(Fluorescent resonance energy transfer)技术原理,构建了包含红色荧光蛋白dsRed1与青色荧光蛋白ECFP的全长cDNA,效应分子Pak1或N-WASP的GTP酶联结区域,信号分子Rac1或Cdc42的全长cDNA的几种单分子探针。转染NIH3T3或Hela细胞,以胰岛素、缓激肽为诱导剂,分别激活Rac1、Cdc42信号转导通路。离体荧光光谱检测表明,在两种转染动物细胞中均产生了FRET现象。不同信号转导通路的FRET效率,在诱导激活5min后,均达到最高值,但增加幅度有显著差异。随着诱导时间的延长,FRET效率下降,但下降速率在不同信号转导通路间差异显著。Rac1、Cdc42激活试验证实,诱导激活的转染细胞中,Rac1、Cdc42均处于激活状态(GTP-bound),其在不同诱导时间的相对激活程度与FRET效率表现相同。诱导激活的Rac1、Cdc42信号转导通路,分别导致了转染活细胞中片状伪足、线状伪足的产生。这表明,采用这些单分子探针,可直接监测激活的信号转导通路,在活细胞中的3D时空分布变化图像及其产生的细胞学效应。采用这些单分子探针,分析、判断了一些调节蛋白对Rac1、Cdc42的GEF或GAP特性,从而提供一种可大大简化现有的鉴定待测蛋白分子的方法。

Rho GTPases, including Racl, Cdc42, play a critical role in the regulation of a variety of cellular processes such as cell morphology, cell migration, transcriptional activation and gene expression. We constructed several FRET-based single-molecule probes containing red fluorescent protein dsRedl, cyan fluorescent protein ECFP, the GTPase binding domain of the effector, Pakl or N-WASP, and Racl or Cdc42. Racl and Cdc42 signaling pathways were activated in transfected ceils by the inducer, insulin or bradykinin respectively. In vitro fluorescent spectroscopy assays showed that FRET phenomena were observed in transfected NIH3T3 and Hela ceils. For all 3 signaling pathways in NIH3T3 cells, the values of FRET efficiency reached the highest after induction for 5 rain, but the increasing extents of the values of FRET efficiency varied in 3 signaling pathways. The values of FRET efficiency decreased with the extention of the induction time, but differed significantly in the decreasing speed for the signaling pathways. Racl and Cdc42 activation assays indicated that Racl and Cdc42 were in the activated state （GTP-bound） in the induced cells. Their relative activated activities in the cells induced for different time were consistent with the values of FRET efficiency. The activated Racl, Cdc42 signaling pathways led to the formation of lamelliopodia and filopodia in the transfected cells respectively. The results showed that these single-molecule probes could be used to directly monitor the spatial and temporal imaging of the induced activation of the signaling pathways in living cells. With these single-molecule probes, the GEF or GAP acivities of putative regulatory proteins for Racl and Cdc42 were analyzed and judged, thus greatly simplifying the currently-used methods for identifying the regulatory proteins for Rho GTPases.