Activation of Rac and Cdc42 video imaged by fluorescent resonance energy transfer-based single-molecule probes in the membrane of living cells
Activation of Rac and Cdc42 video imaged by fluorescent resonance energy transfer-based single-molecule probes in the membrane of living cells
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DOI：
10.1128/mcb.22.18.6582-6591.2002
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发表时间：
2002-09-01
影响因子：
5.3
通讯作者：
Matsuda, M
中科院分区：
文献类型：
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作者：
Itoh, RE;Kurokawa, K;Matsuda, M
Rho家族G蛋白，包括Rac和Cdc42，调节多种细胞功能，如形态、运动性和基因表达。我们开发了基于荧光共振能量转移的探针，其可监测膜上Rac1和Cdc42的鸟嘌呤核苷酸交换因子和GTP酶激活蛋白活性之间的局部平衡。这些探针，名为Raichu - Rac和Raichu - Cdc42，由Pak的Cdc42和Rac结合结构域、Rac1或Cdc42、一对绿色荧光蛋白突变体以及Ki - Ras的CAAX盒组成。利用这些探针，我们对Rac和Cdc42的活性进行了视频成像。在运动的HT1080细胞中，Rac和Cdc42的活性朝着前沿逐渐增加，并且当细胞改变方向时迅速降低。在更高的放大倍数下，我们观察到Rac活性在前沿之后紧邻处最高，而Cdc42活性在前沿尖端最为显著。Raichu - Rac和Raichu - Cdc42还被应用于一种快速且简单的测定中，用于分析活细胞中假定的鸟嘌呤核苷酸交换因子（GEFs）和GTP酶激活蛋白（GAPs）。在六种假定的GEFs和GAPs中，我们确定KIAA0362/DBS是Rac和Cdc42的一种GEF，KIAA1256是Cdc42的一种GEF，KIAA0053是Rac和Cdc42的一种GAP，以及KIAA1204是Cdc42的一种GAP。总之，使用这些单分子探针来确定Rac和Cdc42的活性将加速对活细胞中Rac和Cdc42时空调节的分析。
Rho family G proteins, including Rac and Cdc42, regulate a variety of cellular functions such as morphology, motility, and gene expression. We developed fluorescent resonance energy transfer-based probes which monitored the local balance between the activities of guanine nucleotide exchange factors and GTPase-activating proteins for Rac1 and Cdc42 at the membrane. These probes, named Raichu-Rac and Raichu-Cdc42, consisted of a Cdc42- and Rac-binding domain of Pak, Rac1 or Cdc42, a pair of green fluorescent protein mutants, and a CAAX box of Ki-Ras. With these probes, we video imaged the Rac and Cdc42 activities. In motile HT1080 cells, activities of both Rac and Cdc42 gradually increased toward the leading edge and decreased rapidly when cells changed direction. Under a higher magnification, we observed that Rac activity was highest immediately behind the leading edge, whereas Cdc42 activity was most prominent at the tip of the leading edge. Raichu-Rac and Raichu-Cdc42 were also applied to a rapid and simple assay for the analysis of putative guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) in living cells. Among six putative GEFs and GAPS, we identified KIAA0362/DBS as a GEF for Rac and Cdc42, KIAA1256 as a GEF for Cdc42, KIAA0053 as a GAP for Rac and Cdc42, and KIAA1204 as a GAP for Cdc42. In conclusion, use of these single-molecule probes to determine Rac and Cdc42 activity will accelerate the analysis of the spatiotemporal regulation of Rac and Cdc42 in a living cell.