Centre de Recherche Public-Santé, 84, Val Fleuri, L-1526 Luxembourg, Luxembourg.
Mol Plant. 2013 Mar;6(2):483-502. doi: 10.1093/mp/sss075. Epub 2012 Aug 28.
The two LIM domain-containing proteins from plants (LIMs) typically exhibit a dual cytoplasmic-nuclear distribution, suggesting that, in addition to their previously described roles in actin cytoskeleton organization, they participate in nuclear processes. Using a south-western blot-based screen aimed at identifying factors that bind to plant histone gene promoters, we isolated a positive clone containing the tobacco LIM protein WLIM2 (NtWLIM2) cDNA. Using both green fluorescent protein (GFP) fusion- and immunology-based strategies, we provide clear evidence that NtWLIM2 localizes to the actin cytoskeleton, the nucleus, and the nucleolus. Interestingly, the disruption of the actin cytoskeleton by latrunculin B significantly increases NtWLIM2 nuclear fraction, pinpointing a possible novel cytoskeletal-nuclear crosstalk. Biochemical and electron microscopy experiments reveal the ability of NtWLIM2 to directly bind to actin filaments and to crosslink the latter into thick actin bundles. Electrophoretic mobility shift assays show that NtWLIM2 specifically binds to the conserved octameric cis-elements (Oct) of the Arabidopsis histone H4A748 gene promoter and that this binding largely relies on both LIM domains. Importantly, reporter-based experiments conducted in Arabidopsis and tobacco protoplasts confirm the ability of NtWLIM2 to bind to and activate the H4A748 gene promoter in live cells. Expression studies indicate the constitutive presence of NtWLIM2 mRNA and NtWLIM2 protein during tobacco BY-2 cell proliferation and cell cycle progression, suggesting a role of NtWLIM2 in the activation of basal histone gene expression. Interestingly, both live cell and in vitro data support NtWLIM2 di/oligomerization. We propose that NtWLIM2 functions as an actin-stabilizing protein, which, upon cytoskeleton remodeling, shuttles to the nucleus in order to modify gene expression.
植物中的两个 LIM 结构域蛋白（LIMs）通常表现出双重细胞质-核分布，这表明它们除了先前描述的在肌动蛋白细胞骨架组织中的作用外，还参与核过程。我们使用基于南部印迹的筛选方法，旨在鉴定与植物组蛋白基因启动子结合的因子，分离出一个阳性克隆，其中包含烟草 LIM 蛋白 WLIM2（NtWLIM2）cDNA。我们使用绿色荧光蛋白（GFP）融合和免疫策略，提供了明确的证据表明 NtWLIM2定位于肌动蛋白细胞骨架、核和核仁。有趣的是，Latrunculin B 破坏肌动蛋白细胞骨架会显著增加 NtWLIM2 的核分数，这指出了一种新的细胞骨架-核串扰的可能性。生化和电子显微镜实验揭示了 NtWLIM2 直接结合肌动蛋白丝并将后者交联成厚的肌动蛋白束的能力。电泳迁移率变动分析表明，NtWLIM2特异性结合拟南芥组蛋白 H4A748 基因启动子的保守八聚体顺式元件（Oct），并且这种结合在很大程度上依赖于两个 LIM 结构域。重要的是，在拟南芥和烟草原生质体中进行的基于报告基因的实验证实了 NtWLIM2 结合和激活活细胞中 H4A748 基因启动子的能力。表达研究表明，在烟草 BY-2 细胞增殖和细胞周期进展过程中，NtWLIM2 mRNA 和 NtWLIM2 蛋白持续存在，这表明 NtWLIM2 在基础组蛋白基因表达的激活中发挥作用。有趣的是，活细胞和体外数据均支持 NtWLIM2 的二聚/寡聚化。我们提出 NtWLIM2 作为肌动蛋白稳定蛋白的功能，它在细胞骨架重塑时穿梭到核内以改变基因表达。
