Stimulation of in vivo nuclear transport dynamics of actin and its co-factors IQGAP1 and Rac1 in response to DNA replication stress
Stimulation of in vivo nuclear transport dynamics of actin and its co-factors IQGAP1 and Rac1 in response to DNA replication stress
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DOI：
10.1016/j.bbamcr.2013.06.002
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发表时间：
2013-10-01
影响因子：
5.1
通讯作者：
Henderson, Beric R.
中科院分区：
文献类型：
--
作者：
Johnson, Michael A.;Sharma, Manisha;Henderson, Beric R.
肌动蛋白是细胞骨架的组成部分，在细胞核中参与基因转录、染色质重塑和DNA复制/修复。肌动蛋白通过转运受体输入蛋白-9和输出蛋白-6进出细胞核。在这里，我们通过研究活细胞中的肌动蛋白动力学，解决了细胞周期进程和DNA复制应激对肌动蛋白核定位的影响。首先，我们发现胸腺嘧啶诱导的G1/S期细胞周期阻滞增加了肌动蛋白的细胞核水平以及刺激肌动蛋白聚合的两个因子：IQGAP1和Rac1 GTPase。当细胞暴露于羟基脲诱导DNA复制应激时，肌动蛋白及其调节因子的核定位进一步增强。我们采用活细胞光漂白实验，发现在DNA复制胁迫下，GFP-actin核的进出口率增加了250%。进口速度是出口速度的两倍，这说明了肌动蛋白核积累。更快的穿梭动力学与肌动蛋白的细胞保留减少有关，我们的数据暗示肌动蛋白聚合在核肌动蛋白的应力依赖性摄取中。此外，DNA复制胁迫诱导了IQGAP1和Racl的核转移，并增强了输入动力学。近距离结扎实验表明，IQGAP1在细胞核中与actin和Rac1结合，羟基脲处理后这些复合物的形成增加。我们提出，复制应激检查点触发协调核进入和转运肌动蛋白，以及调节肌动蛋白聚合的因素。(C) 2013 Elsevier B.V.版权所有
Actin, a constituent of the cytoskeleton, is now recognized to function in the nucleus in gene transcription, chromatin remodeling and DNA replication/repair. Actin shuttles in and out of the nucleus through the action of transport receptors importin-9 and exportin-6. Here we have addressed the impact of cell cycle progression and DNA replication stress on actin nuclear localization, through study of actin dynamics in living cells. First, we showed that thymidine-induced G1/S phase cell cycle arrest increased the nuclear levels of actin and of two factors that stimulate actin polymerization: IQGAP1 and Rac1 GTPase. When cells were exposed to hydroxyurea to induce DNA replication stress, the nuclear localization of actin and its regulators was further enhanced. We employed live cell photobleaching assays and discovered that in response to DNA replication stress, GFP-actin nuclear import and export rates increased by up to 250%. The rate of import was twice as fast as export, accounting for actin nuclear accumulation. The faster shuttling dynamics correlated with reduced cellular retention of actin, and our data implicate actin polymerization in the stress-dependent uptake of nuclear actin. Furthermore, DNA replication stress induced a nuclear shift in IQGAP1 and Racl with enhanced import dynamics. Proximity ligation assays revealed that IQGAP1 associates in the nucleus with actin and Rac1, and formation of these complexes increased after hydroxyurea treatment. We propose that the replication stress checkpoint triggers co-ordinated nuclear entry and trafficking of actin, and of factors that regulate actin polymerization. (C) 2013 Elsevier B.V. All rights reserved.