Higashida Chiharu, Suetsugu Shiro, Tsuji Takahiro, Monypenny James, Narumiya Shuh, Watanabe Naoki
Department of Pharmacology, Kyoto University Faculty of Medicine, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
J Cell Sci. 2008 Oct 15;121(Pt 20):3403-12. doi: 10.1242/jcs.030940. Epub 2008 Sep 30.
mDia1 belongs to the formin family of proteins that share FH1 and FH2 domains. Although formins play a critical role in the formation of many actin-based cellular structures, the physiological regulation of formin-mediated actin assembly within the cell is still unknown. Here we show that cells possess an acute actin polymer restoration mechanism involving mDia1. By using single-molecule live-cell imaging, we found that several treatments including low-dose G-actin-sequestering drugs and unpolymerizable actin mutants activate mDia1 to initiate fast directional movement. The FH2 region, the core domain for actin nucleation, is sufficient to respond to latrunculin B (LatB) to increase its actin nucleation frequency. Simulation analysis revealed an unexpected paradoxical effect of LatB that leads to a several fold increase in free G-actin along with an increase in total G-actin. These results indicate that in cells, the actin nucleation frequency of mDia1 is enhanced not only by Rho, but also strongly through increased catalytic efficiency of the FH2 domain. Consistently, frequent actin nucleation by mDia1 was found around sites of vigorous actin disassembly. Another major actin nucleator, the Arp2/3 complex, was not affected by the G-actin increase induced by LatB. Taken together, we propose that transient accumulation of G-actin works as a cue to promote mDia1-catalyzed actin nucleation to execute rapid reassembly of actin filaments.
mDia1属于具有FH1和FH2结构域的formin家族蛋白质。尽管formin在许多基于肌动蛋白的细胞结构形成中起关键作用，但formin介导的细胞内肌动蛋白组装的生理调节仍不清楚。在这里，我们表明细胞拥有一种涉及mDia1的急性肌动蛋白聚合物恢复机制。通过使用单分子活细胞成像，我们发现包括低剂量G-肌动蛋白螯合药物和不可聚合的肌动蛋白突变体在内的几种处理激活mDia1以启动快速定向运动。FH2区域是肌动蛋白成核的核心结构域，足以响应LatB（拉特罗毒素B）以增加其肌动蛋白成核频率。模拟分析揭示了LatB的一种意想不到的矛盾效应，即导致游离G-肌动蛋白增加几倍，同时总G-肌动蛋白也增加。这些结果表明，在细胞中，mDia1的肌动蛋白成核频率不仅通过Rho增强，而且通过FH2结构域催化效率的提高而强烈增强。一致地，在活跃的肌动蛋白解聚位点周围发现mDia1频繁进行肌动蛋白成核。另一个主要的肌动蛋白成核因子Arp2/3复合物不受LatB诱导的G-肌动蛋白增加的影响。综上所述，我们提出G-肌动蛋白的瞬时积累作为一种信号，促进mDia1催化的肌动蛋白成核，以实现肌动蛋白丝的快速重新组装。
