Thomas Clément, Hoffmann Céline, Dieterle Monika, Van Troys Marleen, Ampe Christophe, Steinmetz André
Centre de Recherche Public-Santé, L-1526 Luxembourg.
Plant Cell. 2006 Sep;18(9):2194-206. doi: 10.1105/tpc.106.040956. Epub 2006 Aug 11.
We used confocal microscopy and in vitro analyses to show that Nicotiana tabacum WLIM1, a LIM domain protein related to animal Cys-rich proteins, is a novel actin binding protein in plants. Green fluorescent protein (GFP)-tagged WLIM1 protein accumulated in the nucleus and cytoplasm of tobacco BY2 cells. It associated predominantly with actin cytoskeleton, as demonstrated by colabeling and treatment with actin-depolymerizing latrunculin B. High-speed cosedimentation assays revealed the ability of WLIM1 to bind directly to actin filaments with high affinity. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed a highly dynamic in vivo interaction of WLIM1-GFP with actin filaments. Expression of WLIM1-GFP in BY2 cells significantly delayed depolymerization of the actin cytoskeleton induced by latrunculin B treatment. WLIM1 also stabilized actin filaments in vitro. Importantly, expression of WLIM1-GFP in Nicotiana benthamiana leaves induces significant changes in actin cytoskeleton organization, specifically, fewer and thicker actin bundles than in control cells, suggesting that WLIM1 functions as an actin bundling protein. This hypothesis was confirmed by low-speed cosedimentation assays and direct observation of F-actin bundles that formed in vitro in the presence of WLIM1. Taken together, these data identify WLIM1 as a novel actin binding protein that increases actin cytoskeleton stability by promoting bundling of actin filaments.
我们利用共聚焦显微镜和体外分析表明，烟草WLIM1（一种与动物富含半胱氨酸蛋白相关的LIM结构域蛋白）是植物中一种新型的肌动蛋白结合蛋白。绿色荧光蛋白（GFP）标记的WLIM1蛋白在烟草BY2细胞的细胞核和细胞质中积累。通过共标记和用肌动蛋白解聚剂拉春库林B处理证明，它主要与肌动蛋白细胞骨架相关。高速沉降分析表明WLIM1能够以高亲和力直接结合肌动蛋白丝。光漂白后荧光恢复和光漂白荧光损失显示WLIM1-GFP与肌动蛋白丝在体内存在高度动态相互作用。在BY2细胞中表达WLIM1-GFP显著延迟了拉春库林B处理诱导的肌动蛋白细胞骨架解聚。WLIM1在体外也能稳定肌动蛋白丝。重要的是，在本氏烟草叶片中表达WLIM1-GFP会引起肌动蛋白细胞骨架组织的显著变化，具体而言，与对照细胞相比，肌动蛋白束更少且更粗，这表明WLIM1作为一种肌动蛋白成束蛋白发挥作用。低速沉降分析和对在WLIM1存在下体外形成的F-肌动蛋白束的直接观察证实了这一假设。综上所述，这些数据确定WLIM1是一种新型的肌动蛋白结合蛋白，它通过促进肌动蛋白丝的成束来增加肌动蛋白细胞骨架的稳定性。
