Maisch Jan, Fiserová Jindriska, Fischer Lukás, Nick Peter
Institute of Botany 1, University of Karlsruhe, Kaiserstrasse 2, D-76128 Karlsruhe, Germany.
J Exp Bot. 2009;60(2):603-14. doi: 10.1093/jxb/ern307. Epub 2009 Jan 6.
The polarity of actin is a central determinant of intracellular transport in plant cells. To visualize actin polarity in living plant cells, the tobacco homologue of the actin-related protein 3 (ARP3) was cloned and a fusion with the red fluorescent protein (RFP) was generated. Upon transient expression of these fusions in the tobacco cell line BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2), punctate structures were observed near the nuclear envelope and in the cortical plasma. These dots could be shown to decorate actin filaments by expressing RFP-ARP3 in a marker line, where actin was tagged by GFP (green fluorescent protein)-FABD (fimbrin actin-binding domain 2). When actin filaments were disrupted by latrunculin B or by prolonged cold treatment, and subsequently allowed to recover, the actin filaments reformed from the RFP-ARP3 structures, that therefore represented actin nucleation sites. The intracellular distribution of these sites was followed during the formation of pluricellular files, and it was observed that the density of RFP-ARP3 increased in the apex of the polarized, terminal cells of a file, whereas it was equally distributed in the central cells of a file. These findings are interpreted in terms of position-dependent differences of actin organization.
肌动蛋白的极性是植物细胞内运输的核心决定因素。为了观察活植物细胞中的肌动蛋白极性，克隆了肌动蛋白相关蛋白3（ARP3）的烟草同源物，并将其与红色荧光蛋白（RFP）融合。在烟草细胞系BY-2（烟草品种亮黄2）中瞬时表达这些融合蛋白后，在核膜附近和皮质细胞质中观察到点状结构。通过在标记系中表达RFP-ARP3，可以证明这些点标记了肌动蛋白丝，在该标记系中，肌动蛋白由绿色荧光蛋白（GFP）-FABD（丝束蛋白肌动蛋白结合结构域2）标记。当肌动蛋白丝被拉春库林B或长时间冷处理破坏，随后恢复时，肌动蛋白丝从RFP-ARP3结构重新形成，因此这些结构代表肌动蛋白成核位点。在多细胞列形成过程中追踪这些位点的细胞内分布，观察到RFP-ARP3的密度在列中极化的末端细胞顶端增加，而在列的中央细胞中均匀分布。这些发现根据肌动蛋白组织的位置依赖性差异进行了解释。
