LPA-induced mutually exclusive subcellular localization of active RhoA and Arp2 mRNA revealed by sequential FRET and FISH.
LPA-induced mutually exclusive subcellular localization of active RhoA and Arp2 mRNA revealed by sequential FRET and FISH.
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DOI：
10.1007/s00418-009-0589-x
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发表时间：
2009-07
影响因子：
2.3
通讯作者：
Liu, Gang
中科院分区：
文献类型：
--
作者：
Mingle, Lisa A.;Bonamy, Ghislain;Barroso, Margarida;Liao, Guoning;Liu, Gang
我们之前证明了 Arp2/3 复合物亚基的 mRNA 定位于成纤维细胞中的突起（Mingle 等人，J Cell Sci 118：2425–2433，2005）。然而，调节 Arp2/3 复合物 mRNA 定位的信号通路仍然未知。在这项研究中，我们发现溶血磷脂酸 (LPA) 是 Arp2 mRNA 通过 RhoA-ROCK 途径定位到成纤维细胞突起的有效诱导剂。由于已知 RhoA 在细胞中局部激活，我们试图了解 Rho 的空间激活如何影响 Arp2 mRNA 定位。通过依次进行荧光共振能量转移 (FRET) 和荧光原位杂交 (FISH)，我们在同一细胞中观察到活性 RhoA 和 Arp2 mRNA。 LPA 刺激后，大约两倍于无血清培养基中的细胞显示出活性 RhoA 和 Arp2 mRNA 相互排斥的定位。这些结果证明了 Rho 局部激活在 Arp2 mRNA 定位中的重要性，并为 Rho 如何调节 Arp2/3 复合体 mRNA 定位提供了新的见解。据我们所知，这是第一份将 FRET 和 FISH 结合起来检测同一细胞中局部蛋白活性和 mRNA 的报告。该方法应该很容易用于检测同一细胞中其他基于荧光蛋白的生物传感器和 DNA/RNA。
We previously demonstrated that mRNAs for the subunits of the Arp2/3 complex localize to protrusions in fibroblasts (Mingle et al. in J Cell Sci 118:2425–2433, 2005). However, the signaling pathway that regulates Arp2/3 complex mRNA localization remains unknown. In this study we have identified lysophosphatidic acid (LPA) as a potent inducer of Arp2 mRNA localization to protrusions in fibroblasts via the RhoA-ROCK pathway. As RhoA is known to be activated locally in the cells, we sought to understand how spatial activation of Rho affects Arp2 mRNA localization. By sequentially performing fluorescence resonance energy transfer (FRET) and fluorescence in situ hybridization (FISH), we have visualized active RhoA and Arp2 mRNA in the same cells. Upon LPA stimulation, approximately two times more cells than those in the serum-free medium showed mutually exclusive localization of active RhoA and Arp2 mRNA. These results demonstrate the importance of localized activation of Rho in Arp2 mRNA localization and provide new insights as to how Rho regulates Arp2/3 complex mRNA localization. To our best knowledge, this is the first report in which FRET and FISH are combined to detect localized protein activity and mRNA in the same cells. This method should be easily adopted for the detection of other fluorescence protein based biosensors and DNA/RNA in the same cells.
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